Platform for generating safe cell therapeutics

ABSTRACT

Provided are cytoplasts (enucleated cells), methods for making cytoplasts, compositions comprising cytoplasts, and methods for using cytoplasts.

CLAIM OF PRIORITY

This application is a continuation of U.S. application Ser. No.16/715,859, filed on Dec. 16, 2019, which is a continuation ofInternational Application No. PCT/US2018/045686, filed on Aug. 7, 2018,which claims benefit to U.S. Provisional Patent Application Ser. No.62/542,133, filed Aug. 7, 2017. The entire contents of the foregoing arehereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. CA097022awarded by the National Institutes of Health. The Government has certainrights in the invention.

TECHNICAL FIELD

The present disclosure relates, at least in part, to the field ofbiotechnology, and more specifically, to methods and compositions usingcytoplasts (e.g., enucleated cells) for treatment, prevention ofdisease, prophylactic treatment, adjuvant therapy, or immunomodulationin healthy or diseased subjects.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 1, 2018, isnamed Sequence Listing.txt and is 17.1 MB in size.

BACKGROUND

Current techniques and tools for cell-based therapies are often prone tounwanted and dangerous side effects, such as uncontrolled proliferation,increased mutation rate, and anti-DNA immune responses.

SUMMARY

The present disclosure is based, at least in part, on the generation ofcytoplasts for use as a safe and controllable therapeutic and/ordelivery vehicle. The methods and compositions described herein provideseveral advantages. Methods for enucleating cells to generatecytoplasts, cytoplasts, compositions, and methods of using cytoplastsdescribed can offer several benefits over previous cell-basedtherapeutics. First, the cytoplasts, compositions, and methods describedherein can be safer than previous cell-based therapies. There can, insome embodiments, be a reduced risk of transferring genetic material,including, e.g., nuclear-encoded DNA gene transfer to host. Furtherpotential safety advantages can include one or more of: not respondingto the microenvironment(s) of a subject, not proliferating, and notcontributing to disease progression (e.g., compared to nucleatedmesenchymal stem cells).

Second, the cytoplasts described herein can have a limited or defined(e.g., known, or programmable) life span. Third, the cytoplastsdescribed herein can have a reduced size compared to cells in some othercell-based therapies.

Fourth, the cytoplasts described herein can maintain potency followingcryohibernation or cryopreservation. Cryopreservation includes coolingor freezing, and storing, in the short-term or long-term, biologicalmaterial (e.g., cells, cytoplasts) at very low temperatures (e.g., −80°C. in solid CO₂, −196° C. in liquid nitrogen, etc.). Cryohibernationincludes short-term cooling and storing of biological material (e.g.,cells, cytoplasts) in suspended animation, at non-freezing temperatures,such as, e.g., at 4° C. Cryohibernation of cytoplasts can beadvantageous for one or more of the following reasons: cryohibernationis less labor-intensive than cryopreservation, and cytoplasts that haveundergone cryohibernation can be transported (e.g., shipped).

Fifth, the cytoplasts described herein can be extensively engineered.For example, the cytoplasts can be engineered to produce or express atherapeutic entity, or home to specific sites. Other advantages of thepresently claimed invention are described herein. Accordingly, providedherein are cytoplasts, compositions comprising cytoplasts, methods ofusing cytoplasts, and methods of treating a subject, such as providingbenefits to a healthy or unhealthy subject, or treating or diagnosing adisease or condition (e.g., a cancer or a neoplasm, an infection, aninflammatory condition, a neurological disease (e.g., aneurodegenerative disease), a degenerative disease, an autoimmunedisease, a cardiovascular disease, an ischemic disease, a genetic orinherited disorder, a developmental disorder, an ophthalmologic disease,a skeletal disease, a metabolic disease, a toxicosis, an idiopathiccondition, or two or more thereof, or any disease disclosed herein) in asubject. In some embodiments, methods of treating a subject include:administering to the subject a therapeutically effective amount of acomposition comprising a cytoplast (e.g., a recombinant cytoplast, anycytoplast described herein). In some embodiments, a cytoplastadministered to a subject can produce a therapeutic. In someembodiments, a cytoplast administered to a subject can produce one ormore of: a therapeutic DNA molecule, a therapeutic RNA molecule, atherapeutic protein (e.g., an enzyme, an antibody, an antigen, a toxin,cytokine, a protein hormone, a growth factor, a cell surface receptor,or a vaccine), a therapeutic peptide (e.g., a peptide hormone or anantigen), a small molecule therapeutic (e.g., a steroid, a polyketide,an alkaloid, a toxin, an antibiotic, an antiviral, a colchicine, ataxol, a mitomycin, or emtansine), or a therapeutic gene editing factor.In some embodiments, a cytoplast can be engineered to produce one ormore of: a therapeutic DNA molecule, a therapeutic RNA molecule, atherapeutic protein, a therapeutic peptide, a therapeutic smallmolecule, or a therapeutic gene editing factor. In some embodiments, acytoplast does not need to be engineered to produce one or more of: atherapeutic DNA molecule, a therapeutic RNA molecule, a therapeuticprotein, a therapeutic peptide, a therapeutic small molecule, or atherapeutic gene editing factor. For example, in some embodiments, oneor more of: a therapeutic DNA molecule, a therapeutic RNA molecule, atherapeutic protein, a therapeutic peptide, a small moleculetherapeutic, or a therapeutic gene editing factor can be produced by thecell from which the cytoplast was obtained.

In some embodiments, a therapeutic DNA molecule, a therapeutic RNAmolecule, a therapeutic protein, a therapeutic peptide, a small moleculetherapeutic, or a therapeutic gene editing factor can include atargeting moiety. Non-limiting exemplary targeting moieties that can beproduced by or contained in a cytoplast include chemokine receptors,adhesion molecules, and antigens.

In some embodiments, a cytoplast administered to a subject can contain atherapeutic DNA molecule, a therapeutic RNA molecule, a therapeuticprotein (e.g., an enzyme, an antibody, an antigen, a toxin, cytokine, aprotein hormone, a growth factor, a cell surface receptor, or a vaccine,or any therapeutic protein that is currently available or indevelopment), a therapeutic peptide (e.g., a peptide hormone or anantigen, or any therapeutic peptide that is currently available or indevelopment), a small molecule therapeutic (e.g., a steroid, apolyketide, an alkaloid, a toxin, an antibiotic, an antiviral, ananalgesic, an anticoagulant, an antidepressant, an anticancer drug, anantiepileptic, an antipsychotic, a sedative, a colchicine, a taxol, amitomycin, emtansine, or any small molecule therapeutic that iscurrently available or in development), a therapeutic gene editingfactor, a therapeutic nanoparticle, or another therapeutic agent (e.g.,bacteria, bacterial spores, bacteriophages, bacterial components,viruses (e.g., oncolytic viruses), exosomes, lipids, or ions).Non-limiting examples of oncolytic viruses include Talimogenelaherparepvec, Onyx-015, GL-ONC1, CV706, Voyager-V1, and HSV-1716. Somewild-type viruses also show oncolytic behavior, such as Vaccinia virus,Vesicular stomatitis virus, Poliovirus, Reovirus, Senecavirus, ECHO-7,and Semliki Forest virus.

In some embodiments, a therapeutic DNA molecule, a therapeutic RNAmolecule, a therapeutic, a therapeutic peptide, a small moleculetherapeutic, or a therapeutic gene editing factor is not produced by thecytoplast. In some embodiments, a therapeutic DNA molecule, atherapeutic RNA molecule, a therapeutic, a therapeutic peptide, a smallmolecule therapeutic, or a therapeutic gene editing factor is packagedinside the cytoplast.

In some embodiments, the DNA molecule, the RNA molecule, the protein,the peptide, the small molecule therapeutic, and/or the gene-editingfactor are recombinantly expressed. In some embodiments, the cell fromwhich the cytoplast is derived or obtained is engineered to produce oneor more of the DNA molecule, the RNA molecule, the protein, the peptide,the small molecule therapeutic, and/or the gene-editing factor. In someembodiments, the cell from which the cytoplast is derived or obtained isengineered to stably (e.g., permanently) express one or more of the DNAmolecule, the RNA molecule, the protein, the peptide, the small moleculetherapeutic, and/or the gene-editing factor. In some embodiments, thecell from which the cytoplast is derived or obtained is engineered totransiently express one or more of the DNA molecule, the RNA molecule,the protein, the peptide, the small molecule therapeutic, and/or thegene-editing factor. In some embodiments, the cell from which thecytoplast is derived or obtained is engineered prior to enucleation. Insome embodiments, the cytoplast is engineered to transiently express oneor more of the DNA molecule, the RNA molecule, the protein, the peptide,the small molecule therapeutic, and/or the gene-editing factor (e.g.,engineered following enucleation).

In some embodiments, DNA molecule, the RNA molecule, the protein, thepeptide, the small molecule therapeutic, and/or the gene-editing factorare not naturally expressed (i.e., in the absence of engineering) in thecell from which the cytoplast was derived or obtained (i.e., the DNAmolecule, the RNA molecule, the protein, the peptide, the small moleculetherapeutic, and/or the gene-editing factor are exogenous to thecytoplast). In some embodiments, the DNA molecule, the RNA molecule, theprotein, the peptide, the small molecule therapeutic, and/or thegene-editing factor are not naturally expressed in the subject (i.e.,the DNA molecule, the RNA molecule, the protein, the peptide, the smallmolecule therapeutic, and/or the gene-editing factor are exogenous tothe subject). In some embodiments, the DNA molecule, the RNA molecule,the protein, the peptide, the small molecule therapeutic, and/or thegene-editing factor are not naturally expressed in the subject at theintended site of therapy (e.g., a tumor, or a particular tissue, such asthe brain, the intestine, the lungs, the heart, the liver, the spleen,the pancreas, muscles, eyes, and the like) (i.e., the DNA molecule, theRNA molecule, the protein, the peptide, the small molecule therapeutic,and/or the gene-editing factor are exogenous to the intended site oftherapy).

In some embodiments, the DNA molecule, the RNA molecule, the protein,the peptide, the small molecule therapeutic, and/or the gene-editingfactor are naturally expressed (i.e., in the absence of engineering) inthe cell from which the cytoplast was derived or obtained (i.e., the DNAmolecule, the RNA molecule, the protein, the peptide, the small moleculetherapeutic, and/or the gene-editing factor are innately endogenous tothe cytoplast) (e.g., in the absence of engineering of the cell fromwhich the cytoplast was derived or obtained). In some embodiments, theDNA molecule, the RNA molecule, the protein, the peptide, the smallmolecule therapeutic, and/or the gene-editing factor are naturallyexpressed in the subject (i.e., the DNA molecule, the RNA molecule, theprotein, the peptide, the small molecule therapeutic, and/or thegene-editing factor are endogenous to the subject). In some embodiments,the DNA molecule, the RNA molecule, the protein, the peptide, the smallmolecule therapeutic, and/or the gene-editing factor are naturallyexpressed in the subject at the intended site of therapy (e.g., a tumor,or a particular tissue, such as the brain, the intestine, the lungs, theheart, the liver, the spleen, the pancreas, muscles, eyes, and the like)(i.e., the DNA molecule, the RNA molecule, the protein, the peptide, thesmall molecule therapeutic, and/or the gene-editing factor areendogenous to the intended site of therapy).

In some embodiments, therapeutic, e.g., the DNA molecule, the RNAmolecule, the protein, the peptide, the small molecule therapeutic,and/or the gene-editing factor, is derived from a synthetic cell andloaded into the cytoplast.

In some embodiments, the cytoplast expresses a corrected, a truncated,or a non-mutated version and/or copy of the DNA molecule, the RNAmolecule, the protein, the peptide, the small molecule therapeutic,and/or the gene-editing factor as compared to the cell from which thecytoplast was derived or obtained. In some embodiments, the cytoplast isobtained from any nucleated cell (e.g., a eukaryotic cell, a mammaliancell (e.g., a human cell, or any mammalian cell described herein), aprotozoal cell (e.g., an amoeba cell), an algal cell, a plant cell, afungal cell, an invertebrate cell, a fish cell, an amphibian cell, areptile cell, or a bird cell).

In some embodiments, a cytoplast can produce or contain at least 2(e.g., at least 2, 3, 4, 5, or more) different therapeutic DNAmolecules, therapeutic RNA molecules, therapeutic proteins, therapeuticpeptides, small molecule therapeutics, or therapeutic gene-editingfactors, in any combination. For example, in some embodiments, acytoplast can produce or contain a therapeutic DNA molecule and a smallmolecule therapeutic. For example, in some embodiments, a cytoplast canproduce or contain two different small molecule therapeutics. Forexample, in some embodiments, a cytoplast can produce or contain achemokine receptor (e.g., for targeting) and a small moleculetherapeutic.

In some embodiments, the cytoplast is obtained from an immortalizedcell, a cancer cell (e.g., any cancer cell) a primary (e.g.,host-derived) cell, or a cell line. Any non-immortal cell can beimmortalized using methods known in the art. In some embodiments, thecytoplast can be obtained from a cell autologous to the subject. In someembodiments, the cytoplast can be obtained from a cell allogenic to thesubject. In some embodiments, the cytoplast is obtained from an immunecell. In some embodiments, the cytoplast is obtained from a naturalkiller (NK) cell, a neutrophil, a macrophage, a lymphocyte, afibroblast, an adult stem cell (e.g., hematopoietic stem cell, a mammarystem cell, an intestinal stem cell, mesenchymal stem cell, anendothelial stem cell, a neural stem cell, an olfactory adult stem cell,a neural crest stem cell, a skin stem cell, or a testicular cell), amast cell, a basophil, an eosinophil, or an inducible pluripotent stemcell.

In some embodiments, prior to enucleation, two or more cells (e.g., anyof the cells disclosed herein) are fused by any method disclosed hereinor known in the art. Enucleation of the fusion product can result in acytoplast.

In some embodiments, a first cytoplast is fused to a cell or secondcytoplast. In some embodiments, the cell is any nucleated (e.g., amammalian cell (e.g., a human cell, or any mammalian cell describedherein), a protozoal cell (e.g., an amoeba cell), an algal cell, a plantcell, a fungal cell, an invertebrate cell, a fish cell, an amphibiancell, a reptile cell, or a bird cell). In some embodiments, the secondcell is a synthetic cell. Accordingly, provided are methods of alteringthe behavior of a cell comprising fusing the cell with any of thecytoplasts described herein. Also provided herein are methods comprisingadministering to a subject a therapeutically effective amount of a cellto which a cytoplast has been fused.

In some embodiments, the second cytoplast is derived from the same typeof cell as the first cytoplast. In some embodiments, the secondcytoplast is derived from a different type of cell as the firstcytoplast. In some embodiments, the second cytoplast contains orexpresses at least one therapeutic DNA molecule, therapeutic RNAmolecule, therapeutic protein, therapeutic peptide, small moleculetherapeutic, therapeutic gene editing factor, a therapeuticnanoparticle, or another therapeutic agent that is the same as atherapeutic DNA molecule, therapeutic RNA molecule, therapeutic protein,therapeutic peptide, small molecule therapeutic, therapeutic geneediting factor, a therapeutic nanoparticle contained in or expressed bythe first cytoplast. In some embodiments, the second cytoplast containsor expresses at least one therapeutic DNA molecule, therapeutic RNAmolecule, therapeutic protein, therapeutic peptide, small moleculetherapeutic, therapeutic gene editing factor, a therapeuticnanoparticle, or another therapeutic agent that is different from atherapeutic DNA molecule, therapeutic RNA molecule, therapeutic protein,therapeutic peptide, small molecule therapeutic, therapeutic geneediting factor, a therapeutic nanoparticle contained in or expressed bythe first cytoplast. In some embodiments, a first cytoplast can be fusedto a cell or to a second cytoplast using any method known in the art,for example, electrofusion or viral fusion using viral-based cellsurface peptides.

In some embodiments, the therapeutic RNA molecule is messenger RNA(mRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA),microRNA, long non-coding RNA (lncRNA) or a RNA virus. In someembodiments, the therapeutic DNA molecule is single-stranded DNA,double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNAmolecule or a DNA virus. In some embodiments, the therapeutic protein isa cytokine, a growth factor, a hormone, an antibody, a small-peptidebased drug, or an enzyme. In some embodiments, the cytoplast transientlyexpresses the therapeutic DNA molecule, the therapeutic RNA molecule,the therapeutic protein, the therapeutic peptide, the small moleculetherapeutic, and/or the therapeutic gene editing factor. In someembodiments, the expression of the therapeutic DNA molecule, thetherapeutic RNA molecule, the therapeutic protein, the therapeuticpeptide, the small molecule therapeutic, and/or the therapeutic geneediting factor is inducible. In some embodiments, a nucleated cell ispermanently engineered to express the therapeutic DNA molecule, thetherapeutic RNA molecule, the therapeutic protein, the therapeuticpeptide, the small molecule therapeutic, and/or the therapeutic geneediting factor. In some embodiments, the expression of the therapeuticDNA molecule, the therapeutic RNA molecule, the therapeutic protein, thetherapeutic peptide, the small molecule therapeutic, and/or thetherapeutic gene editing factor. In some embodiments of any of themethods described herein, the cytoplast comprises a therapeutic agent ora nanoparticle. In some embodiments, the therapeutic agent is a smallmolecule or a bacteria or an exosome.

In some embodiments, the method further includes administering to thesubject one or more additional therapies. In some embodiments, the oneor more additional therapies is selected from the group consisting of:cell-based therapy, a small molecule, immuno-therapy, chemotherapy,radiation therapy, gene therapy, and surgery.

Provided herein are isolated cytoplasts (e.g., recombinant cytoplasts,or any cytoplasts described herein) comprising a therapeutic DNAmolecule, a therapeutic RNA molecule, a therapeutic protein, atherapeutic peptide, a small molecule therapeutic, a therapeuticgene-editing factor a therapeutic nanoparticle and/or anothertherapeutic agent. In some embodiments, the therapeutic agent is a drugor chemotherapeutic or a gene editing agent.

Also provided herein are methods of making a recombinant cytoplast(e.g., any cytoplast described herein), the method comprising:enucleating a nucleated cell; and introducing into the enucleated cellan therapeutic DNA molecule, an therapeutic RNA molecule, an therapeuticprotein, an therapeutic peptide, a small molecule therapeutic, atherapeutic gene-editing factor, a therapeutic nanoparticle and/oranother therapeutic agent. In some embodiments, the introducing stepprecedes the enucleating step. In some embodiments, the enucleating stepprecedes the introducing step. In some embodiments, the introducing stepresults in a transient expression of the therapeutic DNA molecule, thetherapeutic RNA molecule, the therapeutic protein, the therapeuticpeptide, the small molecule therapeutic, the therapeutic gene editingfactor, or the other therapeutic agent. In some embodiments, (e.g., whenthe introducing step precedes the enucleation step), the introducingstep results in a permanent expression of the therapeutic DNA molecule,the therapeutic RNA molecule, the therapeutic protein, the therapeuticpeptide, the small molecule therapeutic, the therapeutic gene editingfactor, or the other therapeutic agent In some embodiments, thetherapeutic RNA molecule is messenger RNA (mRNA), short hairpin RNA(shRNA), small interfering RNA (siRNA), microRNA, long non-coding RNA(lncRNA) or a RNA virus. In some embodiments, the therapeutic DNAmolecule is single-stranded DNA, double-stranded DNA, anoligonucleotide, a plasmid, a bacterial DNA molecule or a DNA virus. Insome embodiments, the therapeutic DNA or the therapeutic RNA is a genetherapy. In some embodiments, the therapeutic protein is an enzyme, anantibody, a toxin, cytokine, a protein hormone, a growth factor, or avaccine. In some embodiments, a nucleated cell can be cultured (e.g., ina suspension, as adherent cells, as adherent cells in 3D (e.g., insemi-suspension or other nonadherent methods)) under various conditions(e.g., in a cytokine bath, or under hypoxic conditions) beforeenucleation.

Also provided herein are methods of making a recombinant cytoplast thatinclude: transfecting a nucleated cell with a vector; and enucleatingthe transfected cell.

In some embodiments, the vector is a viral vector (e.g., a retrovirusvector (e.g., a lentivirus vector), an adeno-associated virus (AAV)vector, a vesicular virus vector (e.g., vesicular stomatitis virus (VSV)vector), or a hybrid virus vector). In some embodiments, a viral vectorcan be a cytoplasmic-replicating virus. In some embodiments, a viralvector can be a nuclear-replicating virus. In some embodiments,enucleating occurs after the vector integrates into the genome of thenucleated cell. In some embodiments, the vector is transfected after thecell is enucleated. The order of transfection and enucleation can affectthe choice of vector. For example, if transfection occurs beforeenucleation, either a cytoplasmic-replicating virus or anuclear-replicating virus can be an acceptable vector. For example, iftransfection occurs after enucleation, a cytoplasmic-replicating viruscan be a better choice of vector than a nuclear-replicating virus; onthe other hand, a nuclear-replicating virus can be packaged in anenucleated cytoplast to be released upon death of the cytoplast. In someembodiments, the vector comprises a coding sequence of a therapeuticprotein. In some embodiments, the therapeutic protein is an enzyme, anantibody, a toxin, cytokine, a protein hormone, a growth factor, or avaccine.

Also provided herein are methods of treating a subject that include:administering to the subject a therapeutically effective amount of acomposition comprising a cytoplast expressing a therapeutic DNAmolecule, a therapeutic RNA molecule, a therapeutic protein, atherapeutic peptide, a small molecule therapeutic, and/or a therapeuticgene-editing factor. In some embodiments, a method can includeadministering to the subject a therapeutically effective amount of acomposition comprising a naturally derived cytoplast or an engineeredcytoplast expressing a therapeutic DNA molecule, a therapeutic RNAmolecule, a therapeutic protein, a therapeutic peptide, a small moleculetherapeutic, and/or a therapeutic gene-editing factor.

Also provided herein are methods of treating a subject that include:administering to the subject a therapeutically effective amount of acomposition comprising a cytoplast containing a therapeutic DNAmolecule, a therapeutic RNA molecule, a therapeutic protein, atherapeutic peptide, a small molecule therapeutic, a therapeuticgene-editing factor, a therapeutic nanoparticle, and/or anothertherapeutic agent.

In some embodiments, the cytoplast is obtained from a mammalian cell. Insome embodiments, the cytoplast is obtained from an immune cell.

In some embodiments, the composition further includes a targetingmoiety. In some embodiments, the targeting moiety is a cell surfaceprotein.

In some embodiments, the targeting moiety is a secreted protein, or aprotein that is tethered to the extracellular matrix.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of one embodiment of the disclosure inwhich the starting material is either donor-derived allogenic orautologous cells, or engineered cells with a designed function(s).

FIGS. 2A-D: FIG. 2A is a representative fluorescence microscopy image ofcytoplasts produced from human telomerase reverse transcriptase (hTERT)adipose-derived human mesenchymal stem cells (MSC). Cells were stainedwith red dye (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine) (CMTMR) and nuclei were stained withVybrant® Dyecycle™ green. Arrows point to normal nucleated cells andarrowheads point to enucleated cytoplasts. Scale bar=50 μm.

FIG. 2B is a representative fluorescence microscopy image of cytoplastsproduced from HL-60 human neutrophil cells (neutrophil). Cells werestained with red dye CMTMR and nuclei were stained with Vybrant®Dyecycle™ green. Arrows point to nucleated cells and arrowheads point toenucleated cytoplasts. Scale bar=50 μm.

FIG. 2C is a representative fluorescence microscopy image of cytoplastsproduced from NIH3T3 mouse fibroblast cells (fibroblast). Cells werestained with red dye CMTMR and nuclei were stained with Vybrant®Dyecycle™ green. Arrows point to nucleated cells and arrowheads point toenucleated cytoplasts. Scale bar=50 μm.

FIG. 2D is a representative fluorescence microscopy image of cytoplastsproduced from human natural killer cell line NKL cells (NK). Cells werestained with green dye 5-chloromethylfluorescein diacetate (CMFDA) andnuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Arrowspoint to nucleated cells and arrowheads point to enucleated cytoplasts.Scale bar=50 μm.

FIG. 3A is a representative graph showing the relative fold change inviable cells or cytoplasts over time.

FIG. 3B is a representative graph showing the viable cells andcytoplasts after recovery from frozen storage (cryopreservation).

FIG. 3C is a representative graph showing the relative viability ofcytoplasts 24 hours after enucleation (fresh cytoplasts) or 24 hoursafter recovery from frozen storage (cryopreserved) followingenucleation, where fresh and cryopreserved cytoplasts are compared tothe viability of cytoplasts 4 hours after enucleation. Mean±SEM; n=10.

FIG. 4 are representative flow cytometry graphs showing the number ofevents counted over the signal strength of the indicated fluorescentantibody/marker (CD90, CD44, CD146, CD166, CD45, and isotype control).Bone marrow MSCs or MSC-derived cytoplasts were stained 24 hours afterenucleation and then analyzed by flow cytometry with FlowJo software.Green (light gray) represents nucleated MSCs and red (dark gray)represents enucleated cytoplasts.

FIGS. 5A-F′: FIG. 5A is a representative confocal microscopy image ofMSC-derived cytoplasts cultured in two-dimensional (2D) glass bottomchambers, stained with rhodamine phalloidin to visualize F-actincytoskeleton and DAPI to visualize the nuclei. Arrows point to stainedcytoskeleton structures. Scale bar=50 μm.

FIG. 5B is a representative confocal microscopy image of MSCs culturedin 2D glass bottom chambers, stained with rhodamine phalloidin tovisualize F-actin cytoskeleton and with DAPI to visualize the nuclei.Arrows point to stained cytoskeleton structures. Arrowheads point tonuclei. Scale bar=50 μm.

FIG. 5C is a representative confocal microscopy image of MSC-derivedcytoplasts cultured in 2D glass bottom chambers, stained withanti-α-Tubulin antibody to visualize the microtubule network and DAPI tovisualize the nuclei. Arrows point to stained cytoskeleton structures.Scale bar=50 μm.

FIG. 5D is a representative confocal microscopy image of MSCs culturedin 2D glass bottom chambers, stained with anti-α-Tubulin antibody tovisualize the microtubule network and with DAPI to visualize the nuclei.Arrows point to stained cytoskeleton structures. Arrowheads point tonuclei. Scale bar=50 μm.

FIG. 5E is a representative confocal microscopy image of MSC-derivedcytoplasts cultured in a three-dimensional (3D)-collagen matrix for 24hours, stained with rhodamine phalloidin to visualize F-actincytoskeleton and DAPI to visualize the nuclei. Scale bar=50 μm.

FIG. 5E′ is a representative confocal microscopy image of 5E, showingthe unmerged DAPI stain to visualize nuclei. Scale bar=50 μm.

FIG. 5F is a representative confocal microscopy image of MSCs culturedin 3D-collagen matrix for 24 hours, stained with rhodamine phalloidin tovisualize F-actin cytoskeleton and DAPI to visualize the nuclei.Arrowheads point to nuclei. Scale bar=50 μm. μm.

FIG. 5F′ is a representative confocal microscopy image of 5F, showingthe umerged DAPI stain to visualize nuclei. Scale bar=50 μm.

FIGS. 6A-D′: FIG. 6A is a representative phase contrast microscopy imageof MSCs cultured in full media for 16 hours, then fixed and stained withCrystal Violet. Arrowheads point to well-defined nanotubes. Scale bar=50μm.

FIG. 6B is a representative confocal microscopy image of MSCs culturedin full media for 16 hours, then fixed and stained with themitochondrial marker anti-apoptosis-inducing factor (AIF) and DAPI tovisualize nuclei. Arrowheads point to well-defined nanotubes withprominent mitochondrial staining. Green (light gray) representsAIF-labeled mitochondria; blue (dark gray) represents DAPI-stainednuclei. Scale bar=50 μm.

FIG. 6C is a representative phase contrast microscopy image ofMSC-derived cytoplasts cultured in full media for 16 hours, then fixedand stained with Crystal Violet. Arrowheads point to well-definednanotubes. Scale bar=50 μm.

FIG. 6C′ is an enlarged image of FIG. 6C. Arrowheads point towell-defined nanotubes. Scale bar=50 μm.

FIG. 6D is a representative confocal microscopy image of MSC-derivedcytoplasts cultured in full media for 16 hours, then fixed and stainedwith the mitochondrial marker anti-AIF and DAPI to visualize nuclei.Arrowheads point to well-defined nanotubes with prominent mitochondrialstaining. Green (light gray) represents AIF-labeled mitochondria; blue(dark gray) represents DAPI-stained nuclei. Scale bar=50 μm.

FIG. 6D′ is an enlarged image of FIG. 6D. Scale bar=50 μm.

FIG. 7A-E′: FIG. 7A is a representative confocal microscopy image ofadipose-derived MSCs stained with the mitochondrial marker anti-AIF(light gray) and DAPI (dark gray ovals). Arrows point to mitochondria.Arrowheads point to nuclei. Scale bar=50 μm.

FIG. 7A′ is a representative confocal image of MSC-derived cytoplastsstained with the mitochondrial marker anti-AIF (light gray) and DAPI(dark gray ovals). Arrows point to mitochondria. Arrowheads point tonuclei. Scale bar=50 μm.

FIG. 7B is a representative confocal microscopy image of adipose-derivedMSCs stained with the lysosomal marker anti-lysosome-associated membraneprotein 1 (LAMP1, light gray) and DAPI (dark gray ovals). Arrows pointto lysosomes. Arrowheads point to nuclei. Scale bar=50 μm.

FIG. 7B′ is a representative confocal microscopy image of MSC-derivedcytoplasts stained with LAMP1 (light gray) and DAPI (dark gray ovals).Arrows point to lysosomes. Arrowheads point to nuclei. Scale bar=50 μm.

FIG. 7C is a representative confocal microscopy image of adipose-derivedMSCs stained with the Golgi marker anti-receptor binding cancer antigenexpressed on SiSo cells (RCAS1, light gray) and DAPI (dark gray ovals).Arrows point to Golgi. Arrowheads point to nuclei. Scale bar=50 μm.

FIG. 7C′ is a representative confocal microscopy image of MSC-derivedcytoplasts stained with the Golgi marker anti-RCAS1 (light gray) andDAPI (dark gray ovals). Arrows point to Golgi. Arrowheads point tonuclei. Scale bar=50 μm.

FIG. 7D is a representative confocal microscopy image of adipose-derivedMSCs stained with the endoplasmic reticulum (ER) marker anti-proteindisulfide isomerase (PDI, light gray) and DAPI (dark gray ovals). Arrowspoint to ER. Arrowheads point to nuclei. Scale bar=50 μm.

FIG. 7D′ is a representative confocal microscopy image of MSC-derivedcytoplasts stained with the endoplasmic reticulum (ER) marker anti-PDI(light gray) and DAPI (dark gray ovals). Arrows point to ER. Arrowheadspoint to nuclei. Scale bar=50 μm.

FIG. 7E is a representative confocal microscopy image of adipose-derivedMSCs stained with the endosomal marker anti-early endosome antigen 1(EEA1, light gray) and DAPI (dark gray ovals). Arrows point tolysosomes. Arrowheads point to nuclei. Scale bar=50 μm.

FIG. 7E′ is a representative confocal microscopy image of MSC-derivedcytoplasts stained with the endosomal marker anti-EEA1(light gray) andDAPI (dark gray ovals). Arrows point to lysosomes. Arrowheads point tonuclei. Scale bar=50 μm.

FIG. 8A is a representative bright field microscopy images of MSCs orcytoplasts in Boyden chamber assays that successfully migrated to theundersurface of 8.0 μm porous filters in 3 hours and were then stainedwith Crystal Violet. In the negative control, cells and cytoplastsmigrated in culture media containing 2% FBS in both the upper and lowerchambers. In the stimulated group, the bottom surface of the upperchamber was coated with fibronectin, and 100 ng/mL of stromalcell-derived factor 1 alpha (SDF-1a) was added to the lower chamber.Scale bar=50 μm.

FIG. 8B is a representative bar graph showing the ratio of migratingMSCs or cytoplasts treated as in FIG. 8A (negative or stimulated), whereeach quantity was normalized to the loading control (MSCs or cytoplastsdirectly attached to fibronectin-coated plates). Mean±SEM; n=10.

FIG. 9A-D: FIG. 9A is a representative epifluorescence microscopy imageof MSCs incubated with 100 μM of the cell-permeable peptide (Arg)9-FAM(Fluorescein amidite, light gray) and stained with Hoechst 33342(nuclei, dark gray ovals). Arrows indicate Hoechst-stained nuclei;arrowheads indicate positive (Arg)9-FAM signal.

FIG. 9B is a representative epifluorescence microscopy image ofMSC-derived cytoplasts incubated with 100 μM of the cell-permeablefluorescent peptide (Arg)9-FM (light gray) and stained with Hoechst33342 (nuclei, dark gray ovals). Arrows indicate Hoechst-stained nuclei;arrowheads indicate positive (Arg)9-FAM fluorescence.

FIG. 9C is a representative epifluorescence microscopy image of MSCsincubated with 100 μM of the chemotherapeutic drug doxorubicin (lightgray) and stained with Hoechst 33342 (faint dark gray ovals). Arrowsindicate Hoechst-stained nuclei.

FIG. 9D is a representative epifluorescence microscopy image ofMSC-derived cytoplasts 100 μM of the chemotherapeutic drug doxorubicin(light gray) and stained with Hoechst 33342 (faint dark gray ovals).Arrowheads indicate positive doxorubicin fluorescence.

FIG. 9E is a representative bar graph showing the cell and cytoplastaverage corrected total cell fluorescence per area, which models therelative fluorescence while accounting for the size difference betweencells and cytoplasts. Corrected Total Cell Fluorescence=IntegratedDensity−(Area of selected cell*Mean fluorescence of backgroundreadings). Mean±SEM; n=10.

FIG. 10A is a panel of merged and unmerged confocal microscopy and phasecontrast images of MSCs and MSC-derived cytoplasts incubated withfluorescein isothiocyanate (FITC, bright gray dots) labeled smallinterfering RNA (siRNA) for 24 hours and stained with Hoechst 33342(nuclei, solid gray ovals). Arrowheads indicate positive FITC-labeledsiRNA fluorescence. Arrows indicate nuclei. Scale bar=50 μm.

FIG. 10B is a representative bar graph showing the cell and cytoplastaverage corrected total cell fluorescence per area, which models therelative fluorescence while accounting for the size difference betweencells and cytoplasts. Corrected Total Cell Fluorescence=IntegratedDensity−(Area of selected cell*Mean fluorescence of backgroundreadings).

FIG. 11A are representative merged and unmerged epifluorescencemicroscopy images of MSC and MSC-derived cytoplasts 20 hours aftertransfection with purified enhanced green fluorescent protein messengerRNA (EGFP-mRNA) and stained with Hoechst 33342.

FIG. 11B is a representative bar graph showing the EGFP mRNAtransfection efficiency (percentage of transfected cells out of totalcells) of MSCs or MSC-derived cytoplasts treated as in FIG. 11A.Mean±SEM; n=3.

FIG. 11C is a representative bar graph showing the relative EGFPfluorescence intensity between cells and cytoplasts, accounting fordifference in size. Mean±SEM; MSC group, n=27; MSC-derived cytoplastgroup, n=23. Corrected Total Cell Fluorescence=Integrated Density—(Areaof selected cell*Mean fluorescence of background readings). All data arerepresentative of at least two independent experiments.

FIG. 12A is a representative Western blot of cells treated for 10minutes with either control medium, MSC-conditioned medium (MSC CM),MSC-derived cytoplast-conditioned medium (Cytoplast CM), or 50 ng/mL ofvascular endothelial growth factor (VEGF). Immunoblotting was performedfor protein kinase B (Akt), phosphorylated Akt (p-Akt), extracellularsignal-regulated kinase (Erk), phosphorylated Erk (p-Erk).Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was a loading control.

FIG. 12B is a representative bar graph showing the relative Gaussialuciferase (Gluc) activity in media 48 hours after plating MSCstransfected with Gluc mRNA (MSC-Gluc), non-transfected MSC control cell(MSC), MSC-derived cytoplasts transfected with Gluc mRNA(Cytoplast-Gluc), and non-transfected MSC-derived cytoplasts(Cytoplast).

FIG. 12C is a representative bar graph showing the ratio of RAW 264.7macrophages migrating towards a gradient of the indicated conditionedmedia for 4 hours in a Boyden chamber assay. Migratory cells on thelower membrane surface were stained with Crystal Violet and the numberof cells counted per field and normalized to the loading control (cellsdirectly attached to fibronectin-coated plates). Colony stimulatingfactor 1 (CSF-1), 40 ng/mL of mouse CSF-1 as a positive control;MSC-media, conditioned media from MSCs; Cytoplast media, conditionedmedia from MSC-derived cytoplasts. Mean±SEM; n=10.

FIG. 13A is a schematic representation of an interleukin 10 (IL-10) mRNAtransfected into MSC and cytoplasts. Kozak sequence was added in frontof the start codon of the IL-10 mRNA coding region (CDS). 5′UTR and3′UTR of human beta globin (HBB) mRNA were added respectively to the 5′and 3′ end of IL-10 CDS. An artificial 5′Cap was added to the 5′ end ofthe IL-10 mRNA and the pseudouridine modification was engineered toincrease mRNA stability.

FIG. 13B is a bar graph showing IL-10 concentration in the culturemedium of transfected (++) or non-transfected (−−) MSC or MSC-derivedcytoplasts. MSC-derived cytoplasts were transfected with IL-10 mRNA,then seeded in a 24 well plate at 2.5×10⁴ cells/well. Conditioned medium(CM) was collected 24 hours after transfection and the IL-10concentration determined by ELISA.

FIG. 13C is an immunoblot showing protein expression of Stat3 andphosphorylated Stat3 (P-Stat3, a marker of IL-10 activation) inserum-starved RAW macrophage cells treated with the indicatedconditioned media (CM) from MSCs or cytoplasts treated as in FIG. 13Bfor 1 hour. Untreated=no CM treated control. Complete medium=RAW cellstreated with MSC complete culture medium. MSC Ctrl=RAW cells treatedwith CM from non-transfected MSCs. MSC IL-10=RAW cells treated with CMfrom IL-10 mRNA transfected MSCs. Cytoplast Ctrl=RAW cells treated withCM from non-transfected cytoplasts. Cytoplasts IL-10=RAW cells treatedwith CM from IL-10 mRNA transfected cytoplasts.

FIG. 13D is a bar graph showing the concentration of secreted IL-10cytokine in the mouse blood as determined by ELISA. MSC or MSC-derivedcytoplasts were treated as in FIG. 13B and retro-orbitally injected intothe vasculature of C57BL/6 mice. Two hours after injection, animals wereeuthanized, and blood samples were collected by cardiac puncture.Mean±SEM; n=3.

FIG. 14A are representative bright field microscopy images of CrystalViolet-stained MSCs or MSC-derived cytoplasts in a Boyden chamber assaythat invaded to the undersurface of 8.0 μm porous filters coated withBasement Membrane Extract (BME) towards 10% FBS as a chemoattractant for24 hours. Negative=no FBS (negative control). Scale Bar=50 μm.

FIG. 14B is a representative bar graph showing the ratio of MSC orMSC-derived cytoplasts treated as in FIG. 14A that invaded to theundersurface of the membrane compared to the loading control. Mean±SEM;n=18.

FIG. 15A is representative epifluorescence microscopy images (upperpanel) and phase contrast microscopy images (lower panel) of MSCs andcytoplasts in suspension media. Actin cortex was stained with LifeactRFP, while the cell nucleus was stained with Vybrant® Dyecycle™ Green.Arrows point to cytoplasts and arrowhead points to MSC nucleus. Scalebar=20 μm.

FIG. 15B is a representative scatter plot showing the size distributionof MSCs and cytoplasts as measured with Nikon Element software.Mean±SEM; n=80.

FIG. 15C is a representative bar graph showing the detected Vybrant®DiD-labeled MSCs or cytoplasts present in lung. MSCs or cytoplasts werelabeled with DiD dye and retro-orbitally injected into the vasculatureof C57BL/6 mice. Tissues were harvested after 24 hours and cellsuspensions analyzed by flow cytometry. Mean±SEM; n=3.

FIG. 15D is a representative bar graph showing the detected Vybrant® DiDlabeled MSCs or cytoplasts present in liver. Mean±SEM; n=3. MSCs orcytoplasts were labeled with DiD dye and retro-orbitally injected intothe vasculature of C57BL/6 mice. Tissues were harvested after 24 hoursand cell suspensions analyzed by flow cytometry.

FIG. 16A is a schematic of a representative lentivirus vector engineeredto express CXCR4 on MSCs and cytoplasts (SEQ ID NOs. 1-15).

FIG. 16B is a representative flow cytometry graphs showing the number ofevents counted over the signal strength of the cell surface CXCR4expression by fluorescent antibody on engineered cytoplasts andengineered parental MSCs as analyzed by FlowJo.

FIG. 16C is a representative bar graph showing the ratio of migratingcells or cytoplasts that migrated to the undersurface of the Boydenchamber membrane compared to the loading control. Mean±SEM; n=10. MSCsand MSC-derived cytoplasts with and without engineered CXCR4 receptorsas in FIG. 16A were allowed to migrate towards the indicatedconcentrations of SDF-1α for 2 hours in a Boyden chamber assay.

FIG. 17A is a schematic of the lentivirus vector engineered to expressPSGL1 (P-Selectin Glycoprotein Ligand 1) and Fut7 (Fucosyltransferase,glycosylates/activates PSGL1) on MSCs and cytoplasts. The codingsequences of PSGL1 (SEQ ID NO: 16) and Fut7 (SEQ ID NO: 17) were linkedby 2A sequence (SEQ ID NO: 18).

FIG. 17B is a representative flow cytometry graph showing the number ofevents counted over the signal strength of the cell surface PSGL1expression by fluorescent antibody on engineered cytoplasts andengineered parental MSCs as analyzed by FlowJo.

FIG. 17C is a representative graph showing cell surface binding ofP-Selectin with engineered MSCs and MSC-derived cytoplasts as determinedby flow cytometry. MSC control=parental MSCs. Engineered MSC=PSGL1/Fut7engineered MSC. Engineered cytoplast=PSGL1/Fut7 engineered MSC-derivedcytoplasts.

FIG. 18A is a schematic of a lentivirus vector engineered to expressmCD47 (SEQ ID NO: 19) on MSCs and cytoplasts.

FIG. 18B is a representative flow cytometry graph showing the number ofevents counted over the signal strength of the cell surface of mCD47expression on engineered cytoplasts and MSCs as analyzed by FlowJo.

FIG. 18C is a representative bar graph showing the number of livecytoplasts (DiD+) that were not phagocytosed by macrophages (F4/80⁻ andCD11b), indicating that cytoplasts escaped macrophage phagocytosis inthe lung. Mean±SEM; n=3. DiD dye-labeled Control cytoplasts orengineered cytoplasts (mCD47 Cytoplasts) were retro-orbitally injectedinto the vasculature of mice. After 24 hours, tissues were harvested andstained with two different pan-macrophage markers (F4/80 and CD11b).

FIG. 18D is a representative bar graph showing live cytoplasts (DiD+)that were not phagocytosed by macrophages (F4/80⁻ and CD11b⁻),indicating that cytoplasts escaped macrophage phagocytosis in the liver.Mean±SEM; n=3. DiD dye-labeled Control cytoplasts or engineeredcytoplasts (mCD47 Cytoplasts) were retro-orbitally injected into thevasculature of mice. After 24 hours, tissues were harvested and stainedwith two different pan-macrophage markers (F4/80 and CD11b).

FIG. 19A is schematics of IL-12 mRNA design. Kozak sequence was added infront of the start codon of IL-12 mRNA coding region (CDS). 5′UTR and3′UTR of human beta globin (HBB) mRNA were added respectively to the 5′and 3′ end of IL-12 CDS. Artificial 5′Cap was added to the 5′ end of theIL-12 mRNA and the pseudouridine modification was implemented toincrease mRNA stability.

FIG. 19B is a representative line graph showing the secreted IL-12concentration over time in conditioned media of MSCs or MSC-derivedcytoplasts transfected with IL-12 mRNA and then plated at 2.5×10⁴cells/well of 24-well plate. CM was collected at the indicated timepoints and the secreted IL-12 concentration determined by ELISA. MSConly=CM from non-transfected control. MSC IL-12=CM from MSC transfectedwith IL-12 mRNA. Cytoplast IL-12=Cytoplasts transfected with IL-12 mRNA.

FIG. 19C is an immunoblot showing the activation of thephosphorylated/activated form of Stat4 (P-Stat4). Mouse splenocyte cellswere treated with full media, purified IL-12 protein standard or theindicated CM collected from MSCs or cytoplasts engineered as in FIG. 19Bfor 30 minutes. MSC full medium=mouse splenocytes treated with MSCcomplete culture medium. MSC IL-12=treated with CM from IL-12 mRNAtransfected MSCs. Cytoplast IL-12=treated with CM from IL-12mRNA-transfected cytoplasts.

FIG. 19D is a representative scatter plot showing the concentration ofsecreted IL-12 cytokine per mg of tumor protein. IL-12 engineered orcontrol cytoplasts treated as in FIG. 19B were injected into establishedE0771 (mouse medullary breast carcinoma) tumors growing in syngeneicC57BL/6 mice. Forty-eight hours after tumor injection, animals wereeuthanized and tumor samples were collected, lysed and analyzed byELISA. PBS=samples from mice injected with PBS. Cytoplasts=samples frommice injected with non-engineered cytoplasts. Cytoplasts IL-12=samplesfrom mice injected with cytoplasts engineered to express IL-12 cytokine.

FIG. 20A is a scatter plot showing the fold change of expression forinterferon-γ mRNAs. MSC-derived cytoplasts engineered to express IL-12or control cytoplasts without IL-12 were injected into established E0771tumors growing in syngeneic C57BL/6 mice. Forty-eight hours afterinjection, animals were euthanized and tumor samples were collected,lysed and analyzed by Real-time RT-PCR. PBS=samples from mice injectedwith PBS. Cytoplasts=samples from mice injected with non-engineeredcytoplasts. Cytoplasts IL-12=tumor samples from mice injected withcytoplasts engineered to express IL-12 cytokine. Each dot represents amouse tumor sample. Mean±SEM; n=5.

FIG. 20B is a scatter plot showing the fold change of expression forPD-L1 mRNAs. MSC-derived cytoplasts engineered to express IL-12 orcontrol cytoplasts without IL-12 were injected into established E0771tumors growing in syngeneic C57BL/6 mice. Forty-eight hours afterinjection, animals were euthanized and tumor samples were collected,lysed and analyzed by Real-time RT-PCR. PBS=samples from mice injectedwith PBS. Cytoplasts=samples from mice injected with non-engineeredcytoplasts. Cytoplasts IL-12=tumor samples from mice injected withcytoplasts engineered to express IL-12 cytokine. Each dot represents amouse tumor sample. Mean±SEM; n=5.

FIG. 20C is a scatter plot showing the fold change of expression forCXCL9 mRNAs. MSC-derived cytoplasts engineered to express IL-12 orcontrol cytoplasts without IL-12 were injected into established E0771tumors growing in syngeneic C57BL/6 mice. Forty-eight hours afterinjection, animals were euthanized and tumor samples were collected,lysed and analyzed by Real-time RT-PCR. PBS=samples from mice injectedwith PBS. Cytoplasts=samples from mice injected with non-engineeredcytoplasts. Cytoplasts IL-12=tumor samples from mice injected withcytoplasts engineered to express IL-12 cytokine. Each dot represents amouse tumor sample. Mean±SEM; n=5.

FIG. 20D is a bar graph showing the fold change of E0771 subcutaneoustumor sizein C57Bl/6 mice that were injected intratumorally with 3×10⁶IL-12 engineered cytoplasts (Cytoplasts IL12 group) or PBS (PBS group)on day 11, day 14, and day 18 after tumor cell inoculation. The foldchange of tumor size=Tumor volume of day 20/Tumor volume of day 11.Mean±SEM; n=5.

FIG. 21A are epifluorescence microscopy images taken 7 days afterLifeact-RFP expressing MSCs or cytoplasts infected with 0.05 MOI of theoncolytic herpes simplex virus encoding GFP (oHSV-GFP) were injectedinto subcutaneous U87 glioblastoma tumors in nude mice. Arrowheadrepresents RFP-positive cells (indicating MSC survival and proliferationinside the tumor). Arrow represents GFP-positive tumor cells (successfultransfer of oHSV-GFP from MSC or cytoplast to tumor cell). Scale bar=100μm.

FIG. 21B is a bar graph showing percentage of GFP-positive tumor areafor tumors treated as in FIG. 21A, which represents the portion of tumorcells infected by MSCs or cytoplasts carrying the oHSV-GFP virus.

FIG. 21C is a scatter plot showing the ratio of CD8⁺ effector Tlymphocyte cells out of total CD45+(immune cells) present in tumorsinjected with engineered cytoplasts, as analyzed by flow cytometry.Established subcutaneous E0771 tumors in C57Bl/6 mice were injectedintratumorally with oHSV-transfected and IL-12 engineered cytoplasts orPBS only injection (negative control).

FIG. 22A are representative epifluorescence microscopy images showingRFP expression in nucleated MSC cells successfully fused withCre-engineered cytoplasts. MSC-derived cytoplasts were geneticallyengineered to express Cre recombinase, then electrofused at a ratio of3:1 (under 500 V for 100 μs for 3 pulses) with hTERT-MSCs engineered toexpress Loxp-GFP-stop-Loxp-RFP. Fluorescence images were taken aftersorting for RFP and staining with Hoechst. Arrowhead indicatesHoechst-stained nucleus, arrow indicates positive RFP fluorescenceindicating successful Cre-induced expression of RFP. Scale bar=100 FIG.22B is a bar graph showing the percentage of RFP+(fused) cells out oftotal cells. Loxp MSC only=single culture ofLoxp-GFP-stop-Loxp-RFP—hTERT-MSCs. Cre Cytoplasts only=single culture ofcytoplasts engineered to express Cre recombinase.Co-culture=Co-culturing of Loxp MSCs and Cre cytoplasts for 48 hours.Fusion=Electrofusion of Loxp MSC and Cre cytoplasts. Mean±SEM; n=4.

FIG. 23 is a schematic of the lentivirus vector engineered to expressmCCR2 (SEQ ID NO: 20) on MSCs and cytoplasts.

FIG. 24A is a representative scatter plot showing the number ofDiD-labeled MSCs or cytoplasts detected in the lung. MSCs were culturedunder standard adherent conditions (2D) or in suspension by the handingdrop method (3D) to generate 3D cytoplasts. MSCs and cytoplasts werelabeled with Vybrant® DiD dye and retro-orbitally injected into thevasculature of C57BL/6 mice. Tissues were harvested after 24 hours andcell suspensions analyzed by flow cytometry. Mean±SEM; n=2.

FIG. 24B is a representative scatter plot showing the number ofDiD-labeled MSCs or cytoplasts detected in the liver. MSCs were culturedunder standard adherent conditions (2D) or in suspension by the handingdrop method (3D) to generate 3D cytoplasts. MSCs and cytoplasts werelabeled with Vybrant® DiD dye and retro-orbitally injected into thevasculature of C57BL/6 mice. Tissues were harvested after 24 hours andcell suspensions analyzed by flow cytometry. Mean±SEM; n=2.

FIG. 24C is a representative scatter plot showing the number of Vybrant®DiD-labeled MSCs or cytoplasts detected in the spleen. MSCs werecultured under standard adherent conditions (2D) or in suspension by thehanding drop method (3D) to generate 3D cytoplasts. MSCs and cytoplastswere labeled with DiD dye and retro-orbitally injected into thevasculature of C57BL/6 mice. Tissues were harvested after 24 hours andcell suspensions analyzed by flow cytometry. Mean±SEM; n=2.

FIG. 25A is a representative line graph showing the viability of MSC andMSC-derived cytoplasts immediately after recovery from cryohibernationat 4 degrees Celcius for the indicated amounts of time. Viability wasassessed in an automated cell count (Cell Countess) using Trypan bluedye exclusion and displayed as a ratio to the number of input cells.

FIG. 25B is a representative bar graph comparing the migrated MSC andMSC-derived cytoplasts in a Boyden chamber assay immediately afterrecovery from cryohibernation at 4 degrees Celcius for the indicatedamounts of time. Cells and cytoplasts were allowed to migrate for 3hours with either no serum (negative control) or 10% premium FBS (P-FBS)as a chemoattractant in the bottom chamber, and counts were normalizedto loading controls.

DETAILED DESCRIPTION

The present disclosure shows for the first time that cells from whichthe nucleus has been removed (e.g., cytoplasts), as described herein,exhibit therapeutic functions. In some embodiments, cells can be treatedwith cytochalasin B to soften the cortical actin cytoskeleton. Thenucleus can then be physically extracted from the cell body byhigh-speed centrifugation in gradients of Ficoll to generate anucleus-free (enucleated) cytoplast. Because cytoplast and intactnucleated cells sediment to different layers in the Ficoll gradient,cytoplasts can, in some embodiments, be easily isolated and prepared fortherapeutic purposes or fusion to other cells (nucleated or enucleated).The enucleation process can be clinically scalable to process tens ofmillions of cells. Proof of concept data indicate that cytoplasts can beused as a homing vehicle to deliver clinically relevant cargos/payloadsto treat healthy individuals (e.g., to improve energy, recovery fromexercise, or to deliver natural products) or various diseases (e.g., anyof the diseases described herein). For example, cytoplasts may be usedto deliver supplements, anti-aging factors, preventative treatments, andthe like to healthy individuals, e.g., individuals who have not beendiagnosed with a specific disorder for which the delivered therapeuticis effective. Cytoplasts possess significant therapeutic value becausethey can have one or more of the following properties: remain viable forup to 14 days, do not differentiate into other cell types, secretebioactive proteins, can physically migrate/home, can be extensivelyengineered ex vivo to perform specific therapeutic functions, and can befused to the same or other cell types to transfer desirable cellfunctions, natural or engineered. Therefore, cytoplasts may have wideutility as a new cellular vehicle to deliver therapeutically importantbiomolecules, gene editing factors, and disease-targeting cargosincluding chemotherapeutic drugs (e.g., doxorubicin), genes, viruses,bacteria, mRNAs, shRNAs, siRNA, peptides, plasmids and nanoparticles.The present disclosure advantageously enables, in some embodiments, thegeneration of a safe therapeutic, as it is believed that no unwanted DNAis transferred to the subject using the cytoplasts described herein. Insome embodiments, the present disclosure advantageously enablescontrollable therapeutics, as cell death of the cytoplasts can, in someembodiments, occur in a precise amount of time, e.g., 3-4 days. In someembodiments, the cytoplasts described herein can act as a cell-basedcarrier that can be genetically engineered to deliver specific geneediting, disease-fighting, and health promoting cargos to humans oranimals. Finally, manufacturing significant numbers of therapeutic cellsfor clinical applications can be limited and expensive, thereby limitingthe application of many cell-based therapies, especially in the stemcell field. Therefore, it could be beneficial to use immortalized cells(using hTERT, viruses and oncogenes) to increase manufacturingcapabilities, because it can be robust and cost-effective. However,immortalized cells may cause cancer, and thus can be too dangerous fortherapeutic applications. The present disclosure allows for the use oftype of nucleated cell (an immortalized cell, a cancer cell (e.g., anycancer cell) a primary (e.g., host-derived) cell, or a cell line) forlarge-scale manufacturing in culture for therapeutic use, because theycan be rendered safe by enucleation prior to administration or use.

The present disclosure provides methods to produce cell-basedtherapeutics that are safe and controllable in a subject, from anynucleated cell type that maintains a nucleus throughout it lifespan ordoes not naturally enucleate. In some embodiments, the disclosureprovides methods for the removal of the cell nucleus (also calledenucleation) from any nucleated cell derived (e.g., obtained) fromeither normal or cancer cell lines or any primary cell removed from thebody including, but not limited to, commonly used therapeutic cellsderived (e.g., obtained) from the immune system (e.g., natural killer(NK) cells, neutrophils, macrophages, lymphocytes, mast cells,basophils, eosinophils), stem cells (including, for example, iPSC(induced pluripotent stem cells), adult stem cells (e.g., mesenchymalstem cells), and embryonic stem cells), and fibroblasts. Cellenucleation can create a therapeutic cytoplast which is viable for alimited period of time, for example, up to 3-4 days. Therefore, thepresent disclosure, in some aspects, provides a new use for cytoplastsas a safe therapeutic vehicle that cannot perform one or more of thefollowing actions: proliferate, differentiate, permanently engraft intothe subject, become cancerous, or transfer nuclear-encoded DNA/genes tothe subject (e.g., transfer of dangerous nuclear-encoded DNA/genes tothe subject).

For cell-based therapies, FDA approval has, in some cases, rested on theevidence that cells are stable, meaning that they do not change orbecome dangerous once inside a subject. However, current cell products,including primary cells, irradiated cells, or “death-switch” controlledcells, still have the potential to respond to or change in the in vivomicroenvironment. Importantly, current therapies can still retain thepotential to transcribe new genes, which is not a controllable responsein vivo. This gene transcription hampers the ability to satisfyregulatory requirements. In contrast, cytoplasts, which lack a nucleus,generally do not have the potential for new gene transcription even invery different in vivo microenvironments, and therefore are a morecontrolled and safer cell-based therapy.

To date, cell-based therapeutics generally use normal or engineerednucleated cells. Some cell-based therapies irradiate cells prior tosubject administration in order to prevent cell proliferation andinduced lethal DNA-damage. However, this approach induces mutations andproduces significant amounts of reactive oxygen species that canirreversibly damage cellular proteins and DNA, which can release largeamounts of damaged/mutated DNA into the body of a subject. Such productscan be dangerous if they integrate into other cells and/or induce anunwanted anti-DNA immune response. Irradiated cells can also bedangerous because they can transfer their mutated DNA and genes to hostcells by cell-cell fusion. Removing the entire nucleus from a cell is aless damaging and significantly safer method for limiting cellularlifespan that can preclude any introduction of nuclear DNA into asubject. Furthermore, many stem cells, such as mesenchymal stem cells(MSCs), are highly resistant to radiation-induced death, and thereforecannot be rendered safe using this method. In other cases, therapeuticcells have been engineered with a drug-inducible suicide switch to limitcellular lifespan. However, activation of the switch in vivo can requireadministering a subject with potent and potentially harmful drugs withunwanted side effects. While this method can induce suicide in culturecells (e.g., greater than 95%), it is expected to be inefficient whentranslated into the clinic. Without being bound by any particulartheory, it is believed that a drug-inducible suicide switch could be aninsufficient safety measure for clinical practice, since not all cellsin the subject may undergo drug-induced death. Therefore, in the case ofextensively engineered cells or stem cells or cancer cells, adrug-induced suicide switch could be considered dangerous orinsufficient for clinical practice. Moreover, the death of a therapeuticcell can release large amounts of DNA (normal or genetically altered),which can integrate into host cells or induce a dangerous systemicanti-DNA immune response. If the cell mutates and/or loses orinactivates the suicide switch, it can become an uncontrollable mutantcell. In addition, these cells can fuse with host cells in the subject,and therefore transfer DNA (e.g., mutant DNA). Such fused cells can bedangerous because not all host cells inherit the suicide gene, but caninherit some of the therapeutic cell's genes/DNA during chromosomalreorganization and cell hybridization. In addition, for the same reason,therapeutic cells with suicide switches may not be ideal for use as cellfusion partners in vitro. Another method to limit therapeutic celllifespan is heat-induced death that causes severe damage that terminatesbiological functions beneficial in therapeutic use (e.g., proteintranslation). Unlike cytoplasts, nucleated cell therapies and even somecells inactivated by the methods described above can still transfer DNAto the subject since they retain their nucleus and genetic material.Numerous chemicals inhibit cell proliferation and/or cause cell deathprior to therapeutic use, including chemotherapeutic drugs and mitomycinC, etc. However, such drugs can have significant off-target effects thatsignificantly damage the cell, which are unwanted for clinicalapplications due to high toxicities. Many anti-proliferative anddeath-inducing drugs do not effectively inhibit 100% of the cells due toresistance, and unlike cytoplasts, many drug effects are reversible.Thus, this approach is not suitable to prevent cell growth ofimmortalized or cancer cells in vivo.

The present disclosure provides methods for producing therapeuticcytoplasts with either natural or inducible expression and/or uptake ofbiomolecules with therapeutic functions including, but not limited to,DNA/genes (e.g., plasmids) RNA (e.g., mRNA, shRNA, siRNA, miRNA),proteins, peptides, small molecule therapeutics (e.g., small moleculedrugs), gene editing components, nanoparticles, and other therapeuticagents (e.g., bacteria, bacterial spores, bacteriophages, bacterialcomponents, viruses (e.g., oncolytic viruses), exosomes, lipids, orions).

The present disclosure provides methods for the use of cytoplasts as avehicle to deliver therapeutic cargos to subjects including, but notlimited to, DNA/genes (e.g., plasmids), RNA (e.g., mRNA, shRNA, siRNA,miRNA), proteins, peptides, small molecule therapeutics (e.g., smallmolecule drugs), gene editing components, nanoparticles, and othertherapeutic agents (e.g., bacteria, bacterial spores, bacteriophages,bacterial components, viruses (e.g., oncolytic viruses), exosomes,lipids, or ions).

The present disclosure provides methods for the use of cytoplasts toproduce ions, molecules, compounds, complexes, or biomolecules (whichcan be, for example, secreted, intracellular, inducible, or acombination thereof) including, but not limited to, DNA/genes (e.g.,plasmids), RNA (e.g., mRNA, shRNA, siRNA, miRNA), proteins, peptides,small molecule therapeutics (e.g., small molecule drugs), gene editingcomponents, nanoparticles, and other therapeutic agents (e.g., bacteria,bacterial spores, bacteriophages, bacterial components, viruses,exosomes, or lipids).

The present disclosure provides methods for the largescale in vitroproduction of therapeutic cytoplasts derived (e.g., obtained) from anynucleated cell type (e.g., a mammalian cell (e.g., a human cell, or anymammalian cell described herein), a protozoal cell (e.g., an amoebacell), an algal cell, a plant cell, a fungal cell, an invertebrate cell,a fish cell, an amphibian cell, a reptile cell, or a bird cell). Forexample, the cell can have been immortalized and/or oncogenicallytransformed naturally or by genetic engineering.

The present disclosure provides methods for the use of therapeuticcytoplasts (natural or engineered) as fusion partners to other cells orcytoplasts (therapeutic or natural) to enhance and/or transferorganelles and biomolecules (secreted, intracellular, and natural andinducible) including, but not limited to, mitochondria, ribosomes,endosomes, lysosomes, Golgi, DNA/genes (e.g., plasmids), RNA (e.g.,mRNA, shRNA, siRNA, miRNA), proteins (e.g., cytokines, growth factors,and protein hormones), peptides, small molecule therapeutics (e.g.,small molecule drugs), gene editing components, nanoparticles, and othertherapeutic agents (e.g., bacteria, bacterial spores, bacteriophages,bacterial components, viruses (e.g., oncolytic viruses), exosomes,lipids, or ions).

The present disclosure provides methods for the cryopreservation,cryohibernation, storage, and recovery of therapeutic cytoplasts invitro.

The present disclosure provides methods for the use of cytoplasts asbiosensors and signal transduction indicators of biological processesand healthy or disease states.

The present disclosure, in some embodiments, enables the generation of anovel nucleus-free, cell-based product that can be used as a therapeuticand/or can be modified or genetically engineered to deliver specificdisease-fighting and health-promoting cargos to human or animal subjectsin a safe and controllable manner.

Development of effective cell-based therapeutics often requires geneticengineering and the introduction of new genetic material into the genomeof cells ex vivo. However, this process can introduce dangerousmutations into the genome that produce cancer and other life-threateningdiseases, especially if the engineered cells permanently engraft intothe body or fuse with host cells. The present disclosure allows forremoval of the entire nucleus (i.e. all nuclear encoded DNA) from anynucleated cell type for use as a safe therapeutic or as a vehicle todeliver a specific payload, either biological or synthetic in nature.Cytoplasts can be safer than nucleated cells because no nuclear-encodedgenes or foreign or mutant DNA are transferred to the subject, therebycreating unwanted disease states and/or inducing anti-DNA immuneresponses. The present disclosure allows for generation of a newplatform of safe, nuclear-free cell therapeutics derived from anynucleated cell type, either normal or engineered, including, but notlimited to, iPSC (induced pluripotent stem cells), any immortalizedcell, stem cells, primary cells (e.g., host-derived cells), cell lines,any immune cell, or cancerous cells. It is notable that the actualprocess of enucleation was established in the literature more than threedecades ago. However, the use or development of cytoplasts as atherapeutic entity or as a vehicle to deliver any therapeutic cargoeither natural or engineered to a subject has not been demonstrated todate.

A significant problem with many existing cell-based therapeutics is thatafter delivery to the body, the cells proliferate uncontrollably and canpermanently engraft into the body, which can be life-threatening.

Also, the lack of cell control after administration to the subject canmake the delivery of precise doses of therapeutic cells and theirbioactive products difficult (i.e. poor pharmacokinetics). In someembodiments of the present disclosure, cytoplasts can perform many ofthe same biological/therapeutic functions as their nucleatedcounterpart, but do not proliferate or engraft permanently in thesubject, since they can have a defined life span (e.g., of 1 hour to 14days). Thus, the pharmacokinetics of cytoplast-based therapies can bedefinable and significantly safer with controllable and predictableresponses in the subject.

In some embodiments of the present disclosure, cytoplasts can beadministered to a subject beyond their defined life span (e.g. “dead”cytoplasts). For example, the death process of the administeredcytoplasts can have an immunostimulatory effect on the subject.

Prior to patient or subject delivery, traditional cell-basedtherapeutics are commonly modified or genetically altered ex vivo togenerate desirable cellular and therapeutic functions. However, whenthese cells are introduced into the subject, the new host environmentcan significantly reprogram and negatively alter, or otherwise renderthem ineffective. Since cytoplasts are devoid of a nucleus, there is nonew gene transcription, meaning that, in some embodiments, cytoplastscannot respond to reprogramming and detrimental external signals.Therefore, cytoplasts can retain their differentiated phenotype and exvivo engineered therapeutic functions when introduced into the subject,making them a more controllable and predictable therapeutic vehicle.Likewise, normal donor cells that are immediately enucleated can retaintheir in vivo programs/attributes when transplanted into the subject.Overall, cytoplasts can be a more controllable and predictabletherapeutic vehicle than traditional cell-based therapeutics becausethey can retain their in vitro and in vivo phenotypes and biologicalfunctions. Such properties are critically important for many therapeuticapplications that rely on freshly-derived (e.g., freshly-obtained) donorcells or cells engineered ex vivo to perform specific therapeuticfunctions

Unlike nucleated cells, nuclear-free cytoplasts can, in someembodiments, be loaded with high doses of DNA-damaging/gene targetingagents for delivery to subjects as a therapeutic against cancer or otherdiseases. This includes, but is not limited to, DNA-damagingchemotherapeutic drugs, DNA-integrating viruses, oncolytic viruses, andgene therapy applications/delivery including, but not limited to,cluster regularly interspaced short palindromic repeats (CRISPR), smallclusters of cas (CRISPR/Cas system), and plasmids.

In some embodiments, cytoplasts can also innately produce a therapeuticeffect upon administration to a subject, without the loading of anycargo into the cytoplasts. In some embodiments, cytoplasts can betherapeutic without being engineered to produce one or moretherapeutics. In some embodiments, cytoplasts can be therapeutic withneither the loading of cargo into the cytoplast nor being engineered toproduce one or more therapeutics. For example, an unmanipulatedcytoplast itself can have therapeutic properties when delivered into apatient or subject. In some emobodiments, an unmanipulated cytoplast canproduce one or more of: a therapeutic DNA molecule, a therapeutic RNAmolecule, a therapeutic protein, a therapeutic peptide, a small moleculetherapeutic, and/or a gene-editing factor. In some embodiments,unmanipulated cytoplasts (e.g., derived from autologous or allogenicsources) can have the ability to perform one or more of the followingactions: express therapeutic surface proteins, immune stimulatingantigens, or receptors, secrete cytokines, hormones, or proteins,release exosomes, shed membrane particles, be immunostimulatory throughdeath processes or create tunneling nanotubes which can transfermitochondria and other cell-derived biomolecules. In some embodiments, adead cytoplast can innately produce a therapeutic effect.

In some embodiments, cytoplasts can be applied to or cultured with cells(e.g, xenocultured cells) to alter their properties. For example, insome embodiments, cytoplasts (e.g., unmanipulated cytoplasts orengineered cytoplasts) can upregulate health-promoting factors inxenocultured cells, and in some cases, the xenocultured cells can bereturned to the subject from which they were taken.

Unlike nucleated cells, cytoplasts cannot undergo DNA damage-inducedapoptotic death, and therefore can be used in combination withapoptotic-inducing and/or DNA toxic/targeting agents for treatment ofcancer and other diseases.

Cytoplasts are smaller than their nucleated counterparts and for thisreason can migrate better through small openings in the vasculature andtissue parenchyma. In addition, removing the large dense nucleusalleviates a major physical barrier allowing the cell to move freelythrough small openings in the vessels and tissue parenchyma. Therefore,cytoplasts can have improved bio-distribution in the body and movementinto target tissues.

Unlike nucleated cells, the fusion of cytoplasts to the same or anothercell type of similar or different origin generates a unique cell hybridthat lacks problematic nuclear transfer, while maintaining desirabletherapeutic attributes including, but not limited to, cell surfaceproteins, signal transduction molecules, secreted proteins, lipids, andepigenetic changes.

Exosomes and small cellular membrane vesicles derived from therapeuticcells have been shown, in some instances, to possess therapeuticefficacy alone or as delivery vessels, but are markedly different thanand can be limited as compared to cytoplasts. Similarly, red blood cells(RBCs, erythrocytes), have been hypothesized to be useful as drugdelivery systems. RBCs, too, are different from cytoplasts and can havelimitations as compared to cytoplasts. Unlike exosome and membranevesicles and RBCs, cytoplasts can be viable cell-like entities that canretain many active biological processes and all cellular organelles(e.g., ER/Golgi, mitochondrial, endosome, lysosome, cytoskeleton, etc.).Thus, cytoplasts can function like nucleated cells and exhibit criticalbiological functions such as adhesion, tunneling nanotube formation,actin-mediated spreading (2D and 3D), migration, chemoattractantgradient sensing, mitochondrial transfer, mRNA translation, proteinsynthesis, and secretion of exosomes and other bioactive molecules. Oneor more of these functions may not be exhibited by exosomes, smallcellular membrane vesicles, or RBCs. Compared to RBCs, which are derivedfrom erythroblasts, a cytoplast can be derived from any type ofnucleated cell, including, but not limited to iPSC (induced pluripotentstem cells), any immortalized cell, stem cells, primary cells (e.g.,host-derived cells), cell lines, any immune cell, cancerous cells, orfrom any eukaryotic cell.

A limitation to development of cell-based therapeutics for clinical usecan be the inability and inefficiency of producing large enoughquantities of therapeutic cells, especially stem cells. To alleviatethis manufacturing “bottleneck”, immortalized cells (hTERT, viruses, andoncogenes) have been considered for use to increase cell productioncapacity in a cost-effective manner. However, immortalized cells pose ahigh risk for causing cancer, and thus may be too dangerous for in vivotherapeutic purposes, and are not currently approved by the Food andDrug Administration (FDA). Importantly, the present disclosure can allowfor the use of immortalized cells, cancer cells (e.g., any cancer cell),primary (e.g., host-derived) cells, or a cell line for large-scaleproduction of therapeutic cells, because such cells are enucleated priorto delivery to render them a safe therapeutic. The present disclosurealso allows for generating more cells, cell lines, and/or immortalizedcell from individual subjects for large-scale manufacturing andbio-banking for use in autologous or allogenic therapies (see, e.g.,FIG. 1). This can greatly increase the consistency and quality controlof cell-based therapeutics, which can be offered as an off-the-shelfproduct.

Furthermore, cytoplasts can be superior therapeutic vehicles compared tomanmade synthetic nanoparticles and liposome formulations because theyare derived (e.g., obtained) from cells, and thus are fully functioningcell entities (minus the nucleus), therefore exhibiting crucialphysiological functions, cellular attributes/organelles, andphysiological capabilities to produce bioactive molecules with reducedsubject toxicity.

In some embodiments of any of the methods, cytoplasts, and compositionsdescribed herein, a nucleated cell (e.g., an eukaryotic cell, amammalian cell (e.g., a human cell, a canine cell, a feline cell, anequine cell, a porcine cell, a primate cell, a bovine cell, an ovinecell, a rodent cell (e.g., a mouse cell, a guinea pig cell, a hamstercell, or a mouse cell)), an immune cell, or any nucleated cell describedherein), is treated with cytochalasin B to soften the cortical actincytoskeleton. The nucleus is then physically extracted from the cellbody by high-speed centrifugation in gradients of Ficoll to generate anucleus-free cytoplast. As used herein, the term “cytoplast” or“recombinant cytoplast” are used interchangeably and refer to anucleus-free cell that was obtained from a previously nucleated cell(e.g., any cell described herein) that consists of the inner mass of acell and the cell organelles. In some embodiments, a cytoplast canexpress a therapeutic DNA molecule, a therapeutic RNA molecule, atherapeutic protein, a therapeutic peptide, a small moleculetherapeutic, and/or a gene-editing factor. In some embodiments, acytoplast can contain a therapeutic DNA molecule, a therapeutic RNAmolecule, a therapeutic protein, a therapeutic peptide, a small moleculetherapeutic, and/or a gene-editing factor, a nanoparticle, or anothertherapeutic agent. In some embodiments, an empty cytoplast (e.g., acytoplast with no exogenous components) is used as a negative control.

In some embodiments, cytoplasts can be engineered to express, forexample, chemokine receptors, adhesion molecules, antigens, or othermarkers that can improve the homing of the cytoplasts to sites in asubject, or stimulate and/or modulate desired immune reactions. Forexample, a cytoplast can be engineered to express an anti-PD-L1antibody.

In some embodiments, a nucleated cell can be cultured (e.g., in asuspension, as adherent cells, as adherent cells in 3D (e.g., insemi-suspension or other nonadherent methods)) or clonallyselected/expanded before enucleation.

In some embodiments, a cytoplast has a defined life span of less than 1hour to 14 days (e.g., less than 1 hour to 1 hour, less than 1 hour to 6hours, 6 hours to 12 hours, 12 hours to 1 day, 1 day, 2 days, 3 days, 4days, 5, days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 13days, 14 days, 1 to 14 days, 1 to 12 days, 1 to 10 days, 1 to 9 days, 1to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3days, 1 to 2 days, 2 to 14 days, 2 to 12 days, 2 to 10 days, 2 to 8days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3to 14 days, 3 to 12 days, 3 to 10 days, 3 to 8 days, 3 to 7 days, 3 to 6days, 3 to 5 days, 3 to 4 days, 4 to 14 days, 4 to 12 days, 4 to 10days, 4 to 8 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 4 to 7 days, 5to 14 days, 5 to 12 days, 5 to 10 days, 5 to 8 days, 5 to 7 days, 5 to 6days, 6 to 14 days, 6 to 12 days, 6 to 10 days, 6 to 8 days, 6 to 7days, 7 to 14 days, 7 to 12 days, 7 to 10 days, 7 to 8 days, 8 to 14days, 8 to 12 days, 8 to 10 days, 10 to 14 days, 10 to 12 days, 12 to 14days, less than 14 days, less than 12 days, less than 10 days, less than8 days, less than 7 days, less than 6 days, less than 5 days, less than4 days, less than 3 days, less than 2 days, less than 1 day, less than12 hours, or less than 6 hours). In some embodiments, the lifespan of apopulation of cytoplasts can be evaluated by determining the averagetime at which a portion of the cytoplast population (e.g., at least 50%,at least 60% at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% of the population) isdetermined to be dead. Cell death can be determined by any method knownin the art. In some embodiments, the viability of cytoplasts, e.g., atone or more time points, can be evaluated by determining whethermorphometric or functional parameters are intact (e.g. by trypan-bluedye exclusion, evaluating for intact cell membranes, evaluating adhesionto plastics (e.g., in adherent cytoplasts), evaluating cytoplastmigration, negative staining with apoptotic markers, and the like). Insome embodiments, the life span of a cytoplast may be related to thelife span of the cell from which it was obtained. For example, in someembodiments, a cytoplast obtained from a macrophage may live 12 to 24hours.

In some embodiments, a cytoplast is not a naturally occurring enucleatedcell. In some embodiments, a cytoplast is not obtained from a cell thatnaturally undergoes enucleation. In some embodiments, a cytoplast is nota cell that has been enucleated by in the body of a subject. In someembodiments, a cytoplast is not obtained from a cell that would beenucleated by in the body of a subject. In some embodiments, a cytoplastis not obtained from an erythroblast. In some embodiments, a cytoplastis obtained from a cell that maintains a nucleus over its lifespan(e.g., in the absence of manipulations such as enucleation as describedherein). In some embodiments, a cytoplast is not a cell that is found ina subject as an a nucleate cell (e.g., a red blood cell (erythrocyte), aplatelet, a lens cell, or an immediate nucleated precursor thereof). Insome embodiments, a cytoplast includes one or more components selectedfrom the group consisting of an endoplasmic reticulum, a Golgiapparatus, mitochondria, ribosomes, proteasomes, or spliceosomes. Insome embodiments, a cytoplast is characterized by one or more of thefollowing features: adhesion, tunneling nanotube formation,actin-mediated spreading (2D and/or 3D), migration, chemoattractantgradient sensing, mitochondrial transfer, mRNA translation, proteinsynthesis, and secretion of exosomes and/or other bioactive molecules.In some embodiments, a cytoplast is characterized by an ability tosecrete proteins (e.g., using exosomes). In some embodiments, acytoplast has been enucleated ex vivo. In some embodiments, a cytoplasthas been enucleated in vitro. In some embodiments, a cytoplast has beenphysically enucleated (e.g., by centrifugation). In some embodiments, acytoplast is an engineered enucleated cell. In some embodiments, acytoplast is not a red blood cell. In some embodiments, a cytoplast doesnot contain hemoglobin. In some embodiments, a cytoplast does not have abi-concave shape.

In some embodiments, a cytoplast is not obtained from an erythroblast.In some embodiments, a cytoplast is obtained from a cell that would notbecome a red blood cell (erythrocyte). In some embodiments, a cytoplastis obtained from a lymphoid progenitor cell. In some embodiments, acytoplast is obtained from a lymphocyte. In some embodiments, acytoplast is obtained from a mesenchymal stem cell (e.g., from bonemarrow). In some embodiments, a cytoplast is obtained from anendothelial stem cell. In some embodiments, a cytoplast is obtained froma neural stem cell. In some embodiments, a cytoplast is obtained from askin stem cell.

In some embodiments, a cytoplast is at least 1 μm in diameter. In someembodiments, a cytoplast is greater than 1 μm in diameter. In someembodiments, a cytoplast is 1-100 μm in diameter (e.g., 1-90 μm, 1-80μm, 1-70 μm, 1-60 μm, 1-50 μm, 1-40 μm, 1-30 μm, 1-20 μm, 1-10 μm, 1-5μm, 5-90 μm, 5-80 μm, 5-70 μm, 5-60 μm, 5-50 μm, 5-40 μm, 5-30 μm, 5-20μm, 5-10 μm, 10-90 μm, 10-80 μm, 10-70 μm, 10-60 μm, 10-50 μm, 10-40 μm,10-30 μm, 10-20 μm, 10-15 μm 15-90 μm, 15-80 μm, 15-70 μm, 15-60 μm,15-50 μm, 15-40 μm, 15-30 μm, 15-20 μm). In some embodiments, acytoplast is 10-30 μm in diameter. In some embodiments, the diameter ofa cytoplast is between 5-25 μm (e.g., 5-20 μm, 5-15 μm. 5-10 μm, 10-25μm, 10-20 μm, 10-15 μm, 15-25 μm, 15-20 μm, or 20-25 μm). In someembodiments, a cytoplast is not an exosome. Without being bound by anyparticular theory, it is believed that, in some cases, some cytoplastscan advantageously be small enough to allow for better biodistributionor to be less likely to be trapped in the lungs of a subject.

As used herein, the term “eukaryotic cell” refers to a cell having adistinct, membrane-bound nucleus. Such cells may include, for example,mammalian (e.g., rodent, non-human primate, or human), non-mammaliananimal (e.g., fish, bird, reptile, or amphibian), invertebrate, insect,fungal, or plant cells. In some embodiments, the eukaryotic cell is ayeast cell, such as Saccharomyces cerevisiae. In some embodiments, theeukaryotic cell is a higher eukaryote, such as mammalian, avian, plant,or insect cells. In some embodiments, the nucleated cell is a primarycell. In some embodiments, the nucleated cell is an immune cell (e.g., alymphocyte (e.g., a T cell, a B cell), a macrophage, a natural killercell, a neutrophil, a mast cell, a basophil, a dendritic cell, amonocyte, a myeloid-derived suppressor cell, an eosinophil). In someembodiments, the nucleated cell is a phagocyte or a leukocyte. In someembodiments, the nucleated cell is a stem cell (e.g., an adult stem cell(e.g., a hematopoietic stem cell, a mammary stem cell, an intestinalstem cell, mesenchymal stem cell, an endothelial stem cell, a neuralstem cell, an olfactory adult stem cell, a neural crest stem cell, atesticular cell), an embryonic stem cell, an inducible pluripotent stemcell (iPS)). In some embodiments, the nucleated cell is a progenitorcell. In some embodiments, the nucleated cell is from a cell line. Insome embodiments, the nucleated cell is a suspension cell. In someembodiments, the nucleated cell is an adherent cell. In someembodiments, the nucleated cell is a cell that has been immortalized byexpression of an oncogene. In some embodiments, the nucleated cell isimmortalized by the expression of human telomerase reverse transcriptase(hTERT) or any oncogene. In some embodiments, the nucleated cell is apatient or subject derived cell (e.g., an autologous patient-derivedcell, or an allogenic patient-derived cell). In some embodiments, thenucleated cell is transfected with a vector (e.g., a viral vector (e.g.,a retrovirus vector (e.g., a lentivirus vector), an adeno-associatedvirus (AAV) vector, a vesicular virus vector (e.g., vesicular stomatitisvirus (VSV) vector), or a hybrid virus vector), a plasmid) before thenucleated cell is enucleated using any of the enucleation techniquesdescribed herein and known in the art.

Methods of culturing a cell (e.g., any of the cells described herein)are well known in the art. Cells can be maintained in vitro underconditions that favor growth, proliferation, viability, differentiationand/or induction of specific biological functions with therapeuticcapabilities/benefits including, but not limited to, 3-dimensionalculturing, hypoxic environments, culturing on defined extracellularmatrix components, treatment with chemical agents, cytokines, growthfactors or exposure to any exogenous agent natural or synthetic thatinduces a specific desirable cell response.

In some embodiments, cell therapies already used or in development canbe enucleated (e.g., using any of the methods disclosed herein) to formcytoplasts. Non-limiting examples of cell therapies already used or indevelopment include: treatment of cancer using chimeric antigen receptorengineered T cells (CAR-T), NK or macrophages; treatment of inflammatorydiseases including cancer, autoimmune (Crohn's, rheumatoid arthritis,all types of arthritis, and the like), pancreatitis; regenerativemedicine applications, wound healing, bone or cartilage repair, and thelike; treatment of cognitive diseases such as Alzheimer's, Parkinson's,and the like; treatments of graft-vs-host disease; gene therapy (e.g.,for sickle cell anemia, Severe Combined Immunodeficiency(ADA-SCID/X-SCID), cystic fibrosis, hemophilia, Duchenne's musculardystrophy, Huntington's disease, Parkinson's, hypercholesterolemia,Alpha-1 antitrypsin, chronic granulomatous disease, Fanconi anemia, orGaucher Disease); and treatment of infectious diseases, such as, e.g.,HIV, hepatitis, malaria, and the like.

Without wishing to be bound by any particular theory, it is believedthat enucleation of cell therapies already used or in development canpositively affect the safety profile and/or therapeutic benefit of thecell therapy, as, for example, the cytoplasts would be less effected bythe microenvironment of the subject. Further, in some embodiments, suchcytoplasts can be engineered using any of the methods described herein.For example, in some embodiments, such cytoplasts can be engineered toexpress therapeutic DNA molecule, a therapeutic RNA molecule, atherapeutic protein, a therapeutic peptide, a small moleculetherapeutic, and/or a gene-editing factor. In some embodiments, acytoplast can contain a therapeutic DNA molecule, a therapeutic RNAmolecule, a therapeutic protein, a therapeutic peptide, a small moleculetherapeutic, and/or a gene-editing factor, a nanoparticle, or anothertherapeutic agent. In some embodiments, such a cytoplast can beengineered to express, for example, chemokine receptors, adhesionmolecules, antigens, or other markers that can improve the homing of thecytoplasts to sites in a subject, or stimulate and/or modulate desiredimmune reactions. For example, a cytoplast can be engineered to expressan anti-PD-L1 antibody.

In some embodiments of any of the compositions and methods providedherein, the cytoplast is cooled or frozen for later use. Various methodsof preserving cells are known in the art, including, but not limited to,the use of a serum (e.g., Fetal Bovine Serum) and dimethyl sulfoxide(DMSO) at ultralow temperatures (frozen cryopreservation) or hibernationmedia for storage at 4 degrees Celsius (cryohibernation). In someembodiments of any of the compositions and methods provided herein, thecytoplast is thawed prior to use.

Various methods are known in the art that can be used to introduce abiomolecule (e.g., a RNA molecule (e.g., mRNA, miRNA, siRNA, shRNA,lncRNA), a DNA molecule (e.g., a plasmid), a protein, a gene-editingfactor (e.g., a CRISPR/Cas9 gene-editing factor), a peptide, a plasmid)into a cytoplast (e.g., a cytoplast derived from any cell describedherein). Non-limiting examples of methods that can be used to introducea biomolecule into a cytoplast include: electroporation, microinjection,lipofection, transfection, calcium phosphate transfection,dendrimer-based transfection, cationic polymer transfection, cellsqueezing, sonoporation, optical transfection, impalection, hydrodynamicdelivery, magnetofection, and nanoparticle transfection.

In some embodiments of any of the methods and compositions describedherein, introducing further includes expressing the biomolecule in acytoplast. Various expression vectors are known in the art and can beused herein. Non-limiting examples of expression vectors are providedherein. In some embodiments, the expression vector is the vector shownin any one of FIG. 16A, FIG. 17A, FIG. 18A or FIG. 23. Variousgene-editing factors are known in the art. Non-limiting examples ofgene-editing factors include: CRISPR/Cas9 gene-editing, transcriptionactivator-like effector nuclease (TALEN), and zinc finger nucleases.

In some embodiments of any of the compositions and methods providedherein, a therapeutic agent, a virus, an antibody, drug, or ananoparticle is introduced into the cytoplasts. In some embodiments, atherapeutic DNA, a therapeutic RNA, a therapeutic protein (e.g., anenzyme, an antibody, an antigen, a toxin, cytokine, a protein hormone, agrowth factor, a cell surface receptor, or a vaccine, or any therapeuticprotein that is currently available or in development), a therapeuticpeptide (e.g., a peptide hormone or an antigen, or any therapeuticpeptide that is currently available or in development), a small moleculetherapeutic (e.g., steroid, a polyketide, an alkaloid, a toxin, anantibiotic, an antiviral, an analgesic, an anticoagulant, anantidepressant, an anticancer drug, an antiepileptic, an antipsychotic,a sedative, a colchicine, a taxol, a mitomycin, emtansine, or any smallmolecule therapeutic that is currently available or in development), atherapeutic gene editing factor, a therapeutic nanoparticle, or anothertherapeutic agent (e.g., bacteria, bacterial spores, bacteriophages,bacterial components, viruses (e.g., oncolytic viruses), exosomes,lipids, or ions is introduced into the cytoplasts.

In some embodiments, the cytoplasts can be treated (e.g. stimulated withor loaded) with exosomes. In some embodiments, treatment with exosomescan be used to introduce a biomolecule, a therapeutic, a therapeuticpeptide, a small molecule therapeutic, a therapeutic gene editingfactor, a therapeutic nanoparticle, or another therapeutic agent (e.g.,bacteria, bacterial spores, bacteriophages, bacterial components,viruses (e.g., oncolytic viruses), exosomes, lipids, or ions into thecytoplasts. In some embodiments, treatment with exosomes can be used toalter the behavior, signaling, secreted factors, or othercharacteristics of the cytoplasts.

The present methods include the use of cytoplasts for treating a disease(e.g., a cancer/neoplasm, an infection, an inflammatory condition, aneurological disease (e.g., a neurodegenerative disease), a degenerativedisease, an autoimmune disease, a cardiovascular disease, an ischemicdisease, a genetic or inherited disorder, a developmental disorder, anophthalmologic disease, a skeletal disease, a metabolic disease, atoxicosis, an idiopathic condition, or two or more thereof), in asubject.

Non-limiting examples of cancers include: acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), cancer in adolescents,adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma,atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile ductcancer, bladder cancer, bone cancer, brain stem glioma, brain tumor,breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor,unknown primary carcinoma, cardiac tumors, cervical cancer, childhoodcancers, chordoma, chronic lymphocytic leukemia (CLL), chronicmyelogenous leukemia (CML), chronic myeloproliferative neoplasms, coloncancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma,bile duct cancer, ductal carcinoma in situ, embryonal tumors,endometrial cancer, ependymoma, esophageal cancer,esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,fallopian tube cancer, fibrous histiocytoma of bone, gallbladder cancer,gastric cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumors (GIST), germ cell tumor, gestational trophoblasticdisease, glioma, glioblastoma, hairy cell tumor, hairy cell leukemia,head and neck cancer, heart cancer, hepatocellular cancer,histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocularmelanoma, islet cell tumors, pancreatic neuroendocrine tumors, Kaposisarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer,leukemia, lip and oral cavity cancer, liver cancer, lung cancer,lymphoma, macroglobulinemia, malignant fibrous histiocytoma of bone,osteocarcinoma, melanoma, Merkel cell carcinoma, mesothelioma,metastatic squamous neck cancer, midline tract carcinoma, mouth cancer,multiple endocrine neoplasia syndromes, multiple myeloma, mycosisfungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferativeneoplasms, myelogenous leukemia, myeloid leukemia, multiple myeloma,myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer,nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-smallcell lung cancer, oral cancer, oral cavity cancer, lip cancer,oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer,parathyroid cancer, penile cancer, pharyngeal cancer, pheochromosytoma,pituitary cancer, plasma cell neoplasm, pleuropulmonary blastoma,pregnancy and breast cancer, primary central nervous system lymphoma,primary peritoneal cancer, prostate cancer, rectal cancer, renal cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, smallintestine cancer, soft tissue sarcoma, a solid cancer, squamous cellcarcinoma, squamous neck cancer, stomach cancer, T-cell lymphoma,testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroidcancer, transitional cell cancer of the renal pelvis and ureter, unknownprimary carcinoma, urethral cancer, uterine cancer, uterine sarcoma,vaginal cancer, vulvar cancer, and Wilms' tumor. In some embodiments, acancer may be primary (e.g., a primary tumor) or metastatic (e.g., ametastatic tumor).

Non-limiting types of infections include viral infections, bacterialinfections, fungal infections, parasitic infections, and protozoalinfections. Non-limiting examples of infections include Acinetobacterinfections, Actinomycosis, African sleeping sickness (Africantrypanosomiasis), AIDS (Acquired immunodeficiency syndrome), Amebiasis,Anaplasmosis, Angiostrongyliasis, Anisakiasis, Anthrax, Arcanobacteriumhaemolyticum infection, Argentine hemorrhagic fever, Ascariasis,Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereusinfection, Bacterial pneumonia, Bacterial vaginosis, Bacteroidesinfection, Balantidiasis, Bartonellosis, Bavlisascaris infection, BKvirus infection, Black piedra, Blastocystosis, Blastomycosis, Bolivianhemorrhagic fever, Botulism (and Infant botulism), Brazilian hemorrhagicfever, Brucellosis, Bubonic plague, Burkholderia infection, Buruliulcer, Calicivirus infection (Norovirus and Sapovirus),Campylobacteriosis, Candidiasis (Moniliasis; Thrush), Capillariasis,Carrion's disease, Cat-scratch disease, Cellulitis, Chagas Disease(American trypanosomiasis), Chancroid, Chickenpox, Chikungunya,Chlamydia, Chlamydophila pneumoniae c infection (Taiwan acuterespiratory agent or TWAR), Cholera, Chromoblastomycosis,Chytridiomycosis, Clonorchiasis, Clostridum difficile colitis,Coccidioidomycosis, Colorado tick fever (CTF), Common cold (Acute viralrhinopharyngitis; Acute coryza), Creutzfeldt-Jakob disease (CJD),Crimean-Congo hemorrhagic fever (CCHF), Cryptococcosis,Cryptosporidiosis, Cutaneous larva migrans (CLM), Cyclosporiasis,Cysticercosis, Cytomegalovirus infection, Dengue fever, Desmodesmusinfection, Dientamoebiasis, Diphtheria, Diphyllobothriasis,Dracunculiasis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis,Enterobiasis (Pinworm infection), Enterococcus infection, Enterovirusinfection, Epidemic typhus, Erythema infectiosum (Fifth disease),Exanthem subitum (Sixth disease), Fasciolasis, Fasciolopsiasis, Fatalfamilial insomnia (FFI), Filariasis, Food poisoning by Clostridiumperfringens, Free-living amebic infection, Fusobacterium infection, Gasgangrene (Clostridial myonecrosis), Geotrichosis,Gerstmann-Straussler-Scheinker syndrome (GSS), Giardiasis, Glanders,Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group Astreptococcal infection, Group B streptococcal infection, Haemophilusinfection, Hand, foot and mouth disease (HFMD), Hantavirus PulmonarySyndrome (HPS), Heartland virus disease, Helicobacter pylori infection,Hemolytic-uremic syndrome (HUS), Hemorrhagic fever with renal syndrome(HFRS), Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E,Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirusinfection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis(HGA), Human immnunodeficiency virus (HIV) infection, Humanmetapneumovirus infection, Human monocytic ehrlichiosis, Humanpapillomavirus (HPV) infection, Human parainfluenza virus infection,Hymenolepiasis, Epstein-Barr virus infectious mononucleosis (Mono),Influenza (flu), Isosporiasis, Kawasaki disease, Keratitis, Kingellakingae infection, Kuru, Lassa fever, Legionellosis (Legionnaires'disease), Legionellosis (Pontiac fever), Leishmaniasis, Leprosy,Leptospirosis, Listeriosis, Lyme disease (Lyme borreliosis), Lymphaticfilariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria,Marburg hemorrhagic fever (MHF), Measles, Middle East respiratorysyndrome (MERS), Melioidosis (Whitmore's disease), Meningitis,Meningococcal disease, Metagonimiasis, Microsporidiosis, Molluscumcontagiosum (MC), Monkeypox, Mumps, Murine typhus (Endemic typhus),Mycoplasma pneumonia, Mycoplasma genitalium infection, Mycetoma(disambiguation), Myiasis, Neonatal conjunctivitis (Ophthalmianeonatorum), Norovirus (children and babies), (New) VariantCreutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis(River blindness), Opisthorchiasis, Paracoccidioidomycosis (SouthAmerican blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosiscapitis (Head lice), Pediculosis corporis (Body lice), Pediculosis pubis(Pubic lice, Crab lice), Pelvic inflammatory disease (PID), Pertussis(Whooping cough), Plague, Pneumococcal infection, Pneumocystis pneumonia(PCP), Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebicmeningoencephalitis (PAM), Progressive multifocal leukoencephalopathy,Psittacosis, Q fever, Rabies, Relapsing fever, Respiratory syncytialvirus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsialinfection, Rickettsialpox, Rift Valley fever (RVF), Rocky Mountainspotted fever (RMSF), Rotavirus infection, Rubella, Salmonellosis, SARS(Severe Acute Respiratory Syndrome), Scabies, Scarlet fever,Schistosomiasis, Sepsis, Shigellosis (Bacillary dysentery), Shingles(Herpes zoster), Smallpox (Variola), Sporotrichosis, Staphylococcal foodpoisoning, Staphylococcal infection, Strongyloidiasis, Subacutesclerosing panencephalitis, Syphilis, Taeniasis, Tetanus (Lockjaw),Tinea barbae (Barber's itch), Tinea capitis (Ringworm of the Scalp),Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch), Tineamanum (Ringworm of the Hand), Tinea nigra, Tinea pedis (Athlete's foot),Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis versicolor),Toxocariasis (Ocular Larva Migrans (OLM)), Toxocariasis (Visceral LarvaMigrans (VLM)), Toxoplasmosis, Trachoma, Trichinosis, Trichomoniasis,Trichuriasis (Whipworm infection), Tuberculosis, Tularemia, Typhoidfever, Typhus fever, Ureaplasma urealyticum infection, Valley fever,Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Vibriovulnificus infection, Vibrio parahaemolyticus enteritis, Viralpneumonia, West Nile Fever, White piedra (Tinea blanca), Yersniapseudotuberculosis infection, Yersiniosis, Yellow fever, Zika fever, andZygomycosis.

Non-limiting examples of neurological diseases include Amyotrophiclateral sclerosis (ALS), Alzheimer's disease, Bell's palsy, brainaneurysm, brain injury, brain tumor, cerebral palsy, chronic fatiguesyndrome, concussion, dementia, epilepsy, Guillain-Barré syndrome,headache, Huntington's disease migraine, multiple sclerosis, musculardystrophy, Neuralgia, neuropathy, neuromuscular and related diseases,Parkinson's disease, psychiatric conditions (e.g., depression,obsessive-compulsive disorder), scoliosis, seizures, spinal cord injury,spinal deformity, spinal disorder (e.g., subacute combineddegeneration), spine tumor, stroke, and vertigo.

Non-limiting examples of autoimmune diseases include Achalasia,Addison's disease, Adult Still's disease, Agammaglobulinemia, Alopeciaareata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBMnephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmunedysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis,Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmuneoophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmuneretinopathy, Axonal & neuronal neuropathy (AMAN), Baló disease, Behcet'sdisease, Benign mucosal pemphigoid, Bullous pemphigoid, Castlemandisease (CD), Celiac disease, Chagas disease, Chronic inflammatorydemyelinating polyneuropathy (CIDP), Chronic recurrent multifocalosteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or EosinophilicGranulomatosis (EGPA), Cicatricial pemphigoid, Cogan's syndrome, Coldagglutinin disease, Congenital heart block, Coxsackie myocarditis, CRESTsyndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis,Devic's disease (neuromyelitis optica), Diabetes (e.g., Type I diabetes,type II diabetes, gestational diabetes), Discoid lupus, Dressler'ssyndrome, Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilicfasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evanssyndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis(temporal arteritis), Giant cell myocarditis, Glomerulonephritis,Goodpasture's syndrome, Granulomatosis with Polyangiitis, Graves'disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Hemolyticanemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoidgestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa),Hypogammalglobulinemia, IgA Nephropathy, IgG4-related sclerosingdisease, Immune thrombocytopenic purpura (ITP), Inclusion body myositis(IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes(Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease,Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus,Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD),Lupus, Lyme disease chronic, Meniere's disease, Microscopic polyangiitis(MPA), Mixed connective tissue disease (MCTD), Mooren's ulcer,Mucha-Habermann disease, Multifocal Motor Neuropathy (MMN) or MMNCB,Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, NeonatalLupus, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid,Optic neuritis, Palindromic rheumatism (PR), PANDAS, Paraneoplasticcerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria(PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis),Parsonnage-Turner syndrome, Pemphigus, Peripheral neuropathy, Perivenousencephalomyelitis, Pernicious anemia (PA), POEMS syndrome, Polyarteritisnodosa, Polyglandular syndromes type I, II, III, Polymyalgia rheumatica,Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomysyndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis,Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cellaplasia (PRCA), Pyoderma gangrenosum, Raynaud's phenomenon, ReactiveArthritis, Reflex sympathetic dystrophy, Relapsing polychondritis,Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever,Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis,Scleroderma, Sjögren's syndrome, Sperm & testicular autoimmunity, Stiffperson syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac'ssyndrome, Sympathetic ophthalmia (SO), Takayasu's arteritis, Temporalarteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP),Tolosa-Hunt syndrome (THS), Transverse myelitis, Ulcerative colitis(UC), Undifferentiated connective tissue disease (UCTD), Uveitis,Vasculitis, Vitiligo, Vogt-Koyanagi-Harada Disease, and Wegener'sgranulomatosis (or Granulomatosis with Polyangiitis (GPA)).

Non-limiting examples of cardiovascular diseases include acutemyocardial infarction, heart failure, refractory angina, coronary arterydisease, rheumatic heart disease, congenital heart disease, stroke,aortic aneurism and/or dissection, peripheral arterial disease, deepvein thrombosis, pulmonary embolism, tumors of the heart, vasculartumors of the brain, cardiomyopathy, heart valve diseases, andpericardial disease.

Non-limiting examples of ophthalmologic diseases include glaucoma,cataract, macular degeneration, diabetic retinopathy, strabismus,retinal detachment, uveitis, amblyopia, dry eye syndrome, keratitis,macular edema, corneal ulcer, optic neuropathy, cytomegalovirusretinitis, corneal dystrophy, hyphema, trachoma, central serousretinopathy, retinopathy of prematurity, endophthalmitis, Leber'scongenital amaurosis, central retinal artery occlusion, trichiasis,papilledema, Graves' ophthalmopathy, uveal melanoma, branch retinal veinocclusion, choroideremia, and maculopathy.

Non-limiting examples of skeletal diseases includeosteochondrodysplasia, achondroplasia, hypophospatasia, achondrogenesis,thanatrophoric dysplasia, osteomalacia, rickets, osteopenia,osteoporosis, Paget's disease, osteomyelitis, osteolysis, Haju-Cheneysyndrome, hypertrophic pulmonary osteoarthropathy, nonossifying fibroma,pseudarthrosis, fibrous dysplasia, hyperostosis, osteocsclerosis, andpycnodysostosis.

Non-limiting examples of metabolic diseases include cystinuria, Fabrydisease, galactosemia, Gaucher disease (type I), Hartnup disease,homocystinuria, Hunter syndrome, Hurler syndrome, Lesch-Nyhan syndrome,maple syrup urine disease, Maroteaux-Lamy syndrome, Morquio syndrome,Niemann-Pick disease (type A), phenylketonuria, Pompe disease,porphyria, Scheie syndrome, Tay-Sachs disease, tyrosinemia(hepatorenal),von Gierke disease (glycogen storage deficiency type IA), and Wilson'sdisease.

In some embodiments, the subject is in need of, has been determined tobe in need of, or is suspected to be in need of a cytoplast treatment.In some embodiments, the cancer can be, e.g., acute myeloid leukemia,bladder cancer, breast cancer, kidney cancer, melanoma, small cell lungcancer, non-small cell lung cancer, pancreatic cancer, or prostatecancer.

In some embodiments, a cytoplast can be used for diagnosis of a diseaseor condition (e.g., a cancer or a neoplasm, an infection, aninflammatory condition, a neurological disease (e.g., aneurodegenerative disease), a degenerative disease, an autoimmunedisease, a cardiovascular disease, an ischemic disease, a genetic orinherited disorder, a developmental disorder, an ophthalmologic disease,a skeletal disease, a metabolic disease, a toxicosis, an idiopathiccondition, or two or more thereof). Accordingly, provided herein aremethods of diagnosing a subject, or determining the presence or absenceof a disease or condition in a subject, comprising administering to thesubject any of the cytoplasts as described herein (e.g., unmanipulatedcytoplasts, or cytoplasts genetically engineered or loaded exogenouslywith bioreporter molecules, or with inducible bioreporter molecules)that can signify a subject's particular health, disease state,condition, or toxin level. In some embodiments, the cytoplasts and/or amolecule expressed or secreted by, or contained within, the cytoplastcan operate as a bioreporter. Such bioreporters can be used in subjectsor ex vivo. In some embodiments, a sample can be obtained from a subject(e.g., blood, urine, stool, or tissue (e.g., a biopsy)). In someembodiments, the cytoplasts can express or contain, e.g., colorimetric,fluorescent, luminescent, chemiluminescent or electrochemical moleculesthat report a measurable clinical signal. The signal can be proportionalto the concentration of a chemical, physical agent, or a biomolecule(e.g. growth factors, insulin, cancer antigens, immune factors), orproportional to gene transcriptional activity, or protein translationalactivity in a subject or a sample from a subject.

As used herein, the term “subject” refers to any organism. For example,a subject can be a mammal, amphibian, fish, reptile, invertebrate, bird,plant, archaea, fungus, or bacteria. In some embodiments, the subject isa mammal. In some embodiments, the subject may be a rodent (e.g., amouse, a rat, a hamster, a guinea pig), a canine (e.g., a dog), a feline(e.g., a cat), an equine (e.g., a horse), an ovine, a bovine, a porcine,a non-human primate, e.g., a simian (e.g., a monkey), an ape (e.g., agorilla, a chimpanzee, an orangutan, a gibbon), or a human. In someembodiments of any of the methods described herein, the subject isbetween 0 and 120 years old (e.g., between birth and one month (e.g., aneonate), between one month and two years (e.g., an infant), between 2years and 12 years (e.g., a child), between twelve years and sixteenyears (e.g., an adolescent), between 1 and 120 years old, between 1 and115 years old, between 1 and 110 years old, between 1 and 105 years old,between 1 and 100 years old, between 1 and 95 years old, between 1 and90 years old between 1 and 85 years old, between 1 and 80 years old,between 1 and 75 years old, between 1 and 70 years old, between 1 and 65years old, between 1 and 60 years old, between 1 and 50 years old,between 1 and 40 years old, between 1 and 30 years old, between 1 and 25years old, between 1 and 20 years old, between 1 and 15 years old,between 1 and 10 years old, between 5 and 120 years old, between 5 and110 years old, between 5 and 100 years old, between 5 and 90 years old,between 5 and 60 years old, between 5 and 50 years old, between 5 and 40years old, between 5 and 30 years old, between 5 and 20 years old,between 5 and 10 years old, between 10 and 120 years old, between 10 and110 years old, between 10 and 100 years old, between 10 and 90 yearsold, between 10 and 80 years old between 10 and 60 years old, between 10and 50 years old, between 10 and 40 years old, between 10 and 30 yearsold, between 10 and 20 years, between 20 and 120 years old, between 20and 110 years old, between 20 and 100 years old, between 20 and 90 yearsold, between 20 and 70 years old, between 20 and 60 years old, between20 and 50 years old, between 20 and 40 years old, between 20 and 30years old, between 30 and 120 years old, between 30 and 110 years old,between 30 and 100 years old, between 30 and 90 years old, between 30and 70 years old, between 30 and 60 years, between 30 and 50 years old,between 40 and 120 years old, between 40 and 110 years old, between 40and 100 years old, between 40 and 90 years old, between 40 and 80 yearsold, between 40 and 60 years old, between 40 and 50 years old, between50 and 120 years old, between 50 and 110 years old, between 50 and 100years old, between 50 and 90 years old, between 50 and 80 years old,between 50 and 70 years old, between 50 and 60 years old, between 60 and120 years old, between 60 and 110 years old, between 60 and 100 yearsold, between 60 and 90 years old, between 60 and 80 years old, between60 and 70 years old, between 70 and 120 years old, between 70 and 110years old, between 70 and 100 years old, between 70 and 90 years old,between 70 and 80 years old, between 80 and 120 years old, between 80and 110 years old, between 80 and 100 years old, between 80 and 90 yearsold, between 90 and 120 years old, between 90 and 110 years old, between90 and 100 years old, between 100 and 120 years old, or between 110 and120 years old). In some embodiments of any of the methods describedherein, the subject is not yet born, e.g., in utero. In some embodimentsof any of the methods described herein, the subject is at least 1 monthold (e.g., at least 2 years old, at least 12 years old, at least 16years old, or at least 18 years old). Any of the methods describedherein can be used to treat a subject, e.g., a diseased subject (i.e., asubject with a disease, e.g., who has been diagnosed with a disease), oran asymptomatic subject (i.e., a subject who clinically presents ashealthy, or who has not been diagnosed with a disease). As used herein,treating includes “prophylactic treatment” which means reducing theincidence of or preventing (or reducing risk of) a sign or symptom of adisease in a subject at risk for the disease, and “therapeutictreatment”, which means reducing signs or symptoms of a disease,reducing progression of a disease, reducing severity of a disease,re-occurrence in a subject diagnosed with the disease. As used herein,the term “treat” means to ameliorate at least one clinical parameter ofthe disease, and/or to provide benefits (e.g., anti-aging,anti-scarring, wound healing, anti-depressant, anti-inflammatory, weightloss).

As used herein, “disease,” “disorder,” and “condition” refer to anabnormality in a subject or any deviation from a healthy state in asubject. Non-limiting examples of diseases and/or conditions include acancer or a neoplasm, an infection, an inflammatory condition, aneurological disease (e.g., a neurodegenerative disease), a degenerativedisease, an autoimmune disease, a cardiovascular disease, an ischemicdisease, a genetic or inherited disorder, a developmental disorder, anophthalmologic disease, a skeletal disease, a metabolic disease, atoxicosis, or an idiopathic condition.

In some embodiments of any of the methods provided herein, thecomposition is administered at least once (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90,100 times) during a period of time (e.g., every day, every 2 days, twicea week, once a week, every week, three times per month, two times permonth, one time per month, every 2 months, every 3 months, every 4months, every 5 months, every 6 months, every 7 months, every 8 months,every 9 months, every 10 months, every 11 months, once a year). Alsocontemplated are monthly treatments, e.g., administering at least onceper month for at least 1 month (e.g., at least two, at least three, atleast four, at least five, at least six or more months, e.g., 12 or moremonths), and yearly treatments (e.g., administration once a year for oneor more years). Administration can be via any route known in the art,e.g., subcutaneous, intravenous, arterial, ocular, oral, intramuscular,intranasal (e.g., inhalation), intraperitoneal, topical, mucosal,epidural, sublingual, epicutaneous, extra-amniotic, inter-articular,intradermal, intraosseous, intrathecal, intrauterine, intravaginal,intravesical, intravitreal, perivascular, and/or rectal administration,or any combination of known administration methods.

In some embodiments, the death process of cytoplasts can have atherapeutic effect on a subject. For example, in some embodiments, thedeath process of cytoplasts can be immunostimulatory. Accordingly,provided herein are methods of administering cytoplasts to a subject,wherein the death of the cytoplasts has a therapeutic effect on thesubject. In some embodiments, the cytoplasts administered to the subjectare dead. In some embodiments, the cytoplasts administered to thesubject, when administered, have a remaining life span of less than 5days (e.g., less than 4 days, less than 3 days, less than 2 days, lessthan 36 hours, less than 1 day, less than 18 hours, less than 12 hours,less than 6 hours, less than 2 hours, or less than 1 hour).

In some embodiments, cells can be removed from a subject and enucleated.In some embodiments, the cells are engineered (e.g., to produce orcontain a therapeutic DNA molecule, a therapeutic RNA molecule, atherapeutic protein, a therapeutic peptide, a small moleculetherapeutic, a therapeutic gene-editing factor a therapeuticnanoparticle and/or another therapeutic agent) before being enucleated.In some embodiments, cells from a subject are enucleated, and thenengineered (e.g., to produce or contain a therapeutic DNA molecule, atherapeutic RNA molecule, a therapeutic protein, a therapeutic peptide,a small molecule therapeutic, a therapeutic gene-editing factor atherapeutic nanoparticle and/or another therapeutic agent). In someembodiments, the cytoplasts (whether or not they have been engineered)are administered to the subject from which the cells were removed.

In some embodiments, the media in which the cytoplasts were culturedand/or stored (a “conditioned media”) can have a therapeutic benefit. Insome embodiments, the media in which cytoplasts were co-cultured and/orstored (e.g., after enucleation) with cells (a “conditioned media”) canhave a therapeutic benefit. In some embodiments, the media in whichcytoplasts fused with cells were cultured and/or stored with cells (a“conditioned media”) can have a therapeutic benefit.

Accordingly, provided herein are methods of treating, preventing, orprophylactically treating, or promoting health in a subject comprisingadministering to the subject conditioned media. Without being bound byany particular theory, it is believed that, in some embodiments, thetherapeutic benefit of cultured media can be due to the presence in themedia of exosomes (e.g., containing therapeutic protein) secreted by thecytoplasts.

In some embodiments of any of the methods provided herein, thecomposition is administered with one or more additional therapies (e.g.,any drug (e.g., antibiotics, antivirals, anti-inflammatory medications)or chemotherapy (e.g., a chemotherapeutic agent (e.g., doxorubicin,paclitaxel, cyclophosphamide), or any of the small molecule therapeuticsdescribed herein), cell-based therapy, radiation therapy, immunotherapy,a small molecule, an inhibitory nucleic acid (e.g., antisense RNA,antisense DNA, miRNA, siRNA, lncRNA), an exosome-based therapy, genetherapy or surgery).

In some embodiments provided herein, the composition further includesone or more additional therapies (e.g., any drug (e.g., antibiotics,antivirals) or chemotherapy (e.g., a chemotherapeutic agent (e.g.,doxorubicin, paclitaxel, cyclophosphamide)), cell-based therapy,radiation therapy, immunotherapy, a small molecule, an inhibitorynucleic acid (e.g., antisense RNA, antisense DNA, miRNA, siRNA, lncRNA)or surgery).

Also, provided herein are compositions (e.g., pharmaceuticalcompositions) that include a cytoplast (e.g., a cytoplast obtained fromany cell described herein). In some embodiments, the compositions areformulated for different routes of administration (e.g., intravenous,subcutaneous, intramuscular, retro-orbital, intraperitoneal). In someembodiments, the compositions can include a pharmaceutically acceptablecarrier (e.g., phosphate buffered saline).

For the systemic administration of therapeutic cells, there are twomajor problems for their successful homing to the diseased tissues.First, most of the cells may be trapped in the small capillaries in thelung or other tissues, which may also cause serious side effects such aspulmonary embolism. Cytoplasts are, in some embodiments, much smallerthan their parental cells (e.g., about 60% of the diameter of parentalcells and 1/8 the volume) and do not have the rigid nucleus, therefore,cytoplasts can pass better through small capillaries and vessels thantheir parental cells. Second, the specific homing of cells to thediseased tissues can depend on the chemokine receptor signaling such asSDF-1α/CXCR4, CCL2/CCR2, and the adhesion molecules such as PSGL-1. Asshown herein, cytoplasts can be engineered to specifically expressfunctional CXCR4, CCR2 as well as glycosylated PSGL-1, which can greatlypromote the specific homing of the engineered cytoplasts.

In some embodiments, the cytoplasts can further include (e.g. byengineering or from the cell from which they were obtained) a targetingmoiety that is expressed on the cell surface of the cytoplast, e.g.,CXCR4, CCR2 or PSGL-1. Non-limiting examples of cell surface proteinsthat may be expressed on the cell surface of the cytoplastinclude:chemokines such as CXCR4, CCR2, CCR1, CCR5, CXCR7, CXCR2, and CXCR1. Insome embodiments, the cytoplasts can further include (e.g. byengineering or from the cell from which they were obtained) a celltargeting moiety that is secreted by the cytoplasts, or is tethered tothe extracellular matrix, e.g., SDF1α or CCL2. Non-limiting examples ofproteins that may be secreted by the cytoplast for cell homing include:SDF1α, CCL2, CCL3, CCL5, CCL8, CCL1, CXCL9, CXCL10, CCL11 and CXCL12. Insome embodiments, the cytoplasts can further include (e.g. byengineering or from the cell from which they were obtained) a surfacemarker that aids in their evasion of the subject immune system. Forexample, in some embodiments, the cytoplasts can include a CD47 marker.Without being bound by any particular theory, it is believed that a CD47marker helps to prevent the cytoplasts from being phagocytosed bymacrophages. Non-limiting examples of cell-matrix receptors andcell-cell adhesion molecules include integrins, cadherins,glycoproteins, and heparin sulfate proteoglycans. Non-limiting examplesof therapeutic molecules include tumor antigens and immunomodulatorypeptides, polyamines, and ATP.

In some embodiments, the cytoplasts can be stored at a temperaturebetween about −80° C. and about 16° C. (e.g., about −80° C. and about12° C., −80° C. and about 10° C., about −80° C. and about 8° C., about−80° C. and about 6° C., about −80° C. and about 4° C., about −80° C.and about 2° C., about −80° C. and about 0° C., about −80° C. and about−4° C., about −80° C. and about −10° C., about −80° C. and about −16°C., about −80° C. and about −20° C., about −80° C. and about −25° C.,about −80° C. and about −30° C., about −80° C. and about −35° C., about−80° C. and about −40° C., about −80° C. and about −45° C., about −80°C. and about −50° C., about −80° C. and about −55° C., about −80° C. andabout −60° C., about −80° C. and about −65° C., about −80° C. and about−70° C., about −60° C. and about 16° C., about −60° C. and about 12° C.,about −60° C. and about 10° C., about −60° C. and about 8° C., about−60° C. and about 6° C., about −60° C. and about 4° C., about −60° C.and about 2° C., about −60° C. and about 0° C., about −60° C. and about−4° C., about −60° C. and about −10° C., about −60° C. and about −10°C., about −60° C. and about −16° C., about −60° C. and about −20° C.,about −60° C. and about −25° C., about −60° C. and about −30° C., about−60° C. and about −35° C., about −60° C. and about −40° C., about −60°C. and about −50° C., about −50° C. and about 16° C., about −50° C. andabout 12° C., about −50° C. and about 10° C., about −50° C. and about 8°C., about −50° C. and about 6° C., about −50° C. and about 4° C., about−50° C. and about 2° C., about −50° C. and about 0° C., about −50° C.and about −4° C., about −50° C. and about −10° C., about −50° C. andabout −16° C., about −50° C. and about −20° C., about −50° C. and about−30° C., about −50° C. and about −40° C., about −20° C. and about 16°C., about −20° C. and about 12° C., about −20° C. and about 10° C.,about −20° C. and about 8° C., about −20° C. and about 6° C., about −20°C. and about 4° C., about −20° C. and about 2° C.,-about 20° C. andabout 0° C., about −20° C. and about −4° C., about −20° C. and about−10° C., about −20° C. and about −15° C., about −10° C. and about 16°C., about −10° C. and about 12° C., about −10° C. and about 10° C.,about −10° C. and about 8° C., about −10° C. and about 6° C., about −10°C. and about 4° C., about −10° C. and about 2° C., about −10° C. andabout 0° C., about −10° C. and about −4° C., about −10° C. and about −6°C., about −4° C. and about 16° C., about −4° C. and about 10° C., about−4° C. and about 6° C., about −4° C. and about 4° C., about −4° C. andabout 2° C., about −4° C. and about 0° C., about −2° C. and about 16°C., about −2° C. and about 12° C., about −2° C. and about 10° C., about−2° C. and about 6° C., about −2° C. and about 4° C., about −2° C. andabout 2° C., about −2° C. and about 0° C., about 0° C. and about 16° C.,about 0° C. and about 14° C., about 0° C. and about 12° C., about 0° C.and about 10° C., about 0° C. and about 8° C., about 0° C. and about 6°C., about 0° C. and about 4° C., about 2° C. and about 16° C., about 2°C. and about 12° C., about 2° C. and about 10° C., about 2° C. and about8° C., about 2° C. and about 6° C., about 2° C. and about 4° C., about4° C. and about 16° C., about 4° C. and about 12° C., about 4° C. andabout 10° C., about 4° C. and about 8° C., about 4° C. and about 6° C.,about 6° C. and about 16° C., about 6° C. and about 12° C., about 6° C.and about 10° C., about 6° C. and about 8° C., about 8° C. and about 16°C., about 8° C. and about 12° C., about 8° C. and about 10° C., about10° C. and about 16° C., about 10° C. and about 12° C., or about 12° C.and about 16° C.) for about 1 day to about 7 days (e.g., about 1 day toabout 6 days, about 1 day to about 5 days, about 1 day to about 4 days,about 1 day to about 3 days, about 1 day to about 2 days, about 2 daysto about 7 days, about 2 days to about 6 days, about 2 days to about 5days, about 2 days to about 4 days, about 2 days to about 3 days, about3 days to about 7 days, about 3 days to about 6 days, about 3 days toabout 5 days, about 3 days to about 4 days, about 4 days to about 7days, about 4 days to about 6 days, about 4 days to about 5 days, about5 days to about 7 days, about 5 days to about 6 days, or about 6 days toabout 7 days).

Also, provided herein are kits that include any composition describedherein. For example, a kit can include instructions for using any of thecompositions or methods described herein. In some embodiments, the kitscan include at least one dose of any of the compositions describedherein.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made without departing formthe spirit and scope of the invention.

Exemplary Embodiments

-   -   Embodiment 1 is a method comprising:        -   administering to a subject a therapeutically effective            amount of a composition comprising a first cytoplast            expressing or containing one or more molecules selected from            the group consisting of: a therapeutic DNA molecule, a            therapeutic RNA molecule, a therapeutic protein, a            therapeutic peptide, a small molecule therapeutic, and a            therapeutic gene-editing factor.    -   Embodiment 2 is the method of embodiment 1, wherein the first        cytoplast is obtained from a cell selected from the group        consisting of a mammalian cell a protozoal cell, an algal cell,        a plant cell, a fungal cell, an invertebrate cell, a fish cell,        an amphibian cell, a reptile cell, or a bird cell.    -   Embodiment 3 is the method of embodiment 2, wherein the cell is        or is derived from a cell harvested from the subject.    -   Embodiment 4 is the method of any one of embodiments 2 to 3,        wherein the cell is or is derived from a cell line, an        immortalized cell, or a cancer cell.    -   Embodiment 5 is the method of any one of embodiments 1 to 4,        wherein the first cytoplast is obtained from an immune cell.    -   Embodiment 6 is the method of any one of embodiments 1 to 5,        wherein the first cytoplast is obtained from a cell selected        from the group consisting of a natural killer (NK) cell, a        neutrophil, a macrophage, an eosinophil, a basophil, a dendritic        cell, and a lymphocyte.    -   Embodiment 7 is the method of any one of embodiments 1 to 4,        wherein the first cytoplast is obtained from a cell selected        from the group consisting of a hematopoietic stem cell, a        mammary stem cell, an intestinal stem cell, a mesenchymal stem        cell, an endothelial stem cell, a neural stem cell, an olfactory        adult stem cell, a neural crest stem cell, a skin stem cell, a        testicular cell, an embryonic stem cell, a fibroblast, or an        inducible pluripotent stem cell.    -   Embodiment 8 is the method of any one of embodiments 1 to 7,        wherein the first cytoplast is fused to a second cytoplast.    -   Embodiment 9 is the method of embodiment 8, wherein the second        cell is obtained from a cell selected from the group consisting        of a mammalian cell a protozoal cell, an algal cell, a plant        cell, a fungal cell, an invertebrate cell, a fish cell, an        amphibian cell, a reptile cell, or a bird cell.    -   Embodiment 10 is the method of any one of embodiments 1 to 9,        wherein the therapeutic RNA molecule is messenger RNA (mRNA),        short hairpin RNA (shRNA), small interfering RNA (siRNA),        microRNA, long non-coding RNA (lncRNA) or a RNA virus.    -   Embodiment 11 is the method of any one of embodiments 1 to 10,        wherein the therapeutic DNA molecule is single-stranded DNA,        double-stranded DNA, an oligonucleotide, a plasmid, a bacterial        DNA molecule or a DNA virus.    -   Embodiment 12 is the method of any one of embodiments 1 to 11,        wherein the therapeutic protein is an enzyme, an antibody, an        antigen, a toxin, cytokine, a protein hormone, a growth factor,        a cell surface receptor, or a vaccine.    -   Embodiment 13 is the method of embodiment any one of embodiments        1 to 12, wherein the cytoplast transiently expresses the        therapeutic DNA molecule, the therapeutic RNA molecule, the        therapeutic protein, the therapeutic peptide, the small molecule        therapeutic, and/or the therapeutic gene editing factor.    -   Embodiment 14 is the method of any one of embodiments 1 to 12,        wherein the expression of therapeutic DNA molecule, the        therapeutic RNA molecule, the therapeutic protein, the        therapeutic peptide, small molecule therapeutic, and/or the        therapeutic gene editing factor is inducible.    -   Embodiment 15 is the method of any one of embodiments 1 to 14,        wherein the peptidic therapeutic is selected from the group        consisting of a peptide hormone and an antigen.    -   Embodiment 16 is the method of any one of embodiments 1 to 15,        wherein the small molecule therapeutic is selected from the        group consisting of steroid, a polyketide, an alkaloid, a toxin,        an antibiotic, an antiviral, an analgesic, an anticoagulant, an        antidepressant, an anticancer drug, an antiepileptic, an        antipsychotic, a sedative, a colchicine, a taxol, a mitomycin,        emtansine, or any small molecule therapeutic that is currently        available or in development.    -   Embodiment 17 is the method of any one of embodiments 1 to 16,        wherein the cytoplast contains a small molecule therapeutic or a        therapeutic nanoparticle.    -   Embodiment 18 is the method of any one of embodiments 1-16,        wherein the cytoplast contains a therapeutic agent selected from        the group consisting of bacteria, bacterial spores,        bacteriophages, bacterial components, viruses, exosomes, lipids,        and ions.    -   Embodiment 19 is the method of embodiment 18, wherein the        viruses are oncolytic viruses.    -   Embodiment 20 is the method of any one of embodiments 1 to 15 or        17 to 19, wherein the small molecule therapeutic is selected        from the group consisting of an anticancer drug, an antibiotic,        or an antiviral.    -   Embodiment 21 is the method of any one of embodiments 1 to 19,        further comprising administering to the subject one or more        additional therapies.    -   Embodiment 22 is the method of embodiment 20, wherein the one or        more additional therapies is selected from the group consisting        of: cell-based therapy, a small molecule, immuno-therapy,        chemotherapy, radiation therapy, gene therapy, and surgery.    -   Embodiment 23 is the method of any one of embodiments 1 to 21,        wherein the first cytoplast expresses an immune system-evading        moiety.    -   Embodiment 24 is the method of embodiment 23, wherein the        immune-system evading moiety is CD47.    -   Embodiment 25 is the method of any one of embodiments 1 to 24,        wherein the first cytoplast or cell from which the first        cytoplast is obtained has been engineered to express the        therapeutic DNA molecule, the therapeutic RNA molecule, the        therapeutic protein, the therapeutic peptide, the non-peptide        therapeutic, and/or the therapeutic gene editing factor.    -   Embodiment 26 is the method of any one of embodiments 1 to 24,        wherein the first cytoplast or cell from which the first        cytoplast is obtained has not been engineered to express any of        the therapeutic DNA molecule, the therapeutic RNA molecule, the        therapeutic protein, the therapeutic peptide, the non-peptide        therapeutic, and/or the therapeutic gene editing factor.    -   Embodiment 27 is the method of any one of embodiments 1 to 26,        wherein the composition further includes a targeting moiety.    -   Embodiment 28 is the method of embodiment 27, wherein the        targeting moiety is a cell surface protein.    -   Embodiment 29 is the method of embodiment 27, wherein the        targeting moiety is a secreted protein or a protein that is        tethered to the extracellular matrix.    -   Embodiment 30 is the method of any one of embodiments 1 to 26,        wherein the cytoplast further comprise a targeting moiety.    -   Embodiment 31 is the method of embodiment 30, wherein the        targeting moiety is a cell surface protein.    -   Embodiment 32 is the method of embodiment 30, wherein the        targeting moiety is a secreted protein or a protein that is        tethered to the extracellular matrix.    -   Embodiment 33 is a cytoplast comprising at least one therapeutic        agent.    -   Embodiment 34 is the cytoplast of embodiment 33 wherein the        therapeutic agent is a therapeutic DNA molecule, a therapeutic        RNA molecule, a therapeutic protein, a therapeutic peptide, a        small molecule therapeutic, or a therapeutic gene editing        factor.    -   Embodiment 35 is the cytoplast of embodiment 34, wherein the        therapeutic RNA molecule is messenger RNA (mRNA), short hairpin        RNA (shRNA), small interfering RNA (siRNA), microRNA, long        non-coding RNA (lncRNA) or a RNA virus.    -   Embodiment 36 is the cytoplast of any one of embodiments 34 to        35, wherein the therapeutic DNA molecule is single-stranded DNA,        double-stranded DNA, an oligonucleotide, a plasmid, a bacterial        DNA molecule or a DNA virus.    -   Embodiment 37 is the cytoplast of any one of embodiments 34 to        36, wherein the therapeutic protein is an enzyme, an antibody,        an antigen, a toxin, cytokine, a protein hormone, a growth        factor, a cell surface receptor, or a vaccine.    -   Embodiment 38 is the cytoplast of any one of embodiments 34 to        37, wherein the peptidic therapeutic is selected from the group        consisting of a peptide hormone and an antigen.    -   Embodiment 39 is the cytoplast of any one of embodiments 34 to        38, wherein the small molecule therapeutic is selected from the        group consisting of a steroid, a polyketide, an alkaloid, a        toxin, an antibiotic, an antiviral, an analgesic, an        anticoagulant, an antidepressant, an anticancer drug, an        antiepileptic, an antipsychotic, a sedative, a colchicine, a        taxol, a mitomycin, emtansine, or any small molecule therapeutic        that is currently available or in development.    -   Embodiment 40 is the cytoplast of any one of embodiments 33 to        38, wherein the therapeutic agent is selected from the group        consisting of a nanoparticle, bacteria, bacterial spores,        bacteriophages, bacterial components, viruses, exosomes, lipids,        and ions.    -   Embodiment 41 is the cytoplast of embodiment 40, viruses are        oncolytic viruses.    -   Embodiment 42 is the cytoplast of any one of embodiments 33 to        41, wherein the small molecule therapeutic is selected from the        group consisting of an anticancer drug, an antibiotic, or an        antiviral.    -   Embodiment 43 is the cytoplast of any one of embodiments 33 to        43, wherein the cytoplast further comprises an immune        system-evading moiety.    -   Embodiment 44 is the cytoplast of embodiment 43, wherein the        immune system-evading moiety is CD47.    -   Embodiment 45 is a method of making a cytoplast, the method        comprising:        -   introducing into a cell a therapeutic DNA molecule, a            therapeutic RNA molecule, a therapeutic protein, a            therapeutic peptide, a small molecule therapeutic, a            therapeutic gene editing factor, an other therapeutic agent,            and/or a therapeutic nanoparticle; and        -   enucleating the cell.    -   Embodiment 46 is the method of embodiment 45, wherein the        introducing step precedes the enucleating step.    -   Embodiment 47 is the method of embodiment 46, wherein the        introducing step results in a permanent expression of the        therapeutic DNA molecule, the therapeutic RNA molecule, the        therapeutic protein, the therapeutic peptide, the small molecule        therapeutic, and/or the therapeutic gene editing factor.    -   Embodiment 48 is the method of embodiment 45, wherein the        enucleation step precedes the introducing step.    -   Embodiment 49 is the method of any one of embodiments 45, 46, or        48, wherein the introducing step results in a transient        expression of the therapeutic DNA molecule, the therapeutic RNA        molecule, the therapeutic protein, the therapeutic peptide, the        small molecule therapeutic, and/or the therapeutic gene editing        factor.    -   Embodiment 50 is the method of any one of embodiments 45 to 49,        wherein the therapeutic RNA molecule is messenger RNA (mRNA),        short hairpin RNA (shRNA), small interfering RNA (siRNA),        microRNA, long non-coding RNA (lncRNA) or a RNA virus.    -   Embodiment 51 is the method of any one of embodiments 45 to 50,        wherein the therapeutic DNA molecule is single-stranded DNA,        double-stranded DNA, an oligonucleotide, a plasmid, a bacterial        DNA molecule or a DNA virus.    -   Embodiment 52 is the method of any one of embodiments 45 to 51,        wherein the therapeutic protein is an enzyme, an antibody, an        antigen, a toxin, cytokine, a protein hormone, a growth factor,        a cell surface receptor, or a vaccine.    -   Embodiment 53 is the method of any one of embodiments 45 to 52,        wherein the peptidic therapeutic is selected from the group        consisting of a peptide hormone and an antigen.    -   Embodiment 54 is the method of any one of embodiments 45 to 53,        wherein the small molecule therapeutic is selected from the        group consisting of a steroid, a polyketide, an alkaloid, a        toxin, an antibiotic, an antiviral, an analgesic, an        anticoagulant, an antidepressant, an anticancer drug, an        antiepileptic, an antipsychotic, a sedative, a colchicine, a        taxol, a mitomycin, emtansine, or any small molecule therapeutic        that is currently available or in development.    -   Embodiment 55 is the method of any one of embodiments 45 to 54,        wherein the cytoplast further comprises a therapeutic        nanoparticle.    -   Embodiment 56 is the method of any one of embodiments 45 to 54,        wherein the cytoplast further comprises a therapeutic agent        selected from the group consisting of bacteria, bacterial        spores, bacteriophages, bacterial components, viruses, exosomes,        lipids, and ions.    -   Embodiment 57 is the method of embodiment 56, wherein the        viruses are oncolytic viruses.    -   Embodiment 58 is the method of any one of embodiments 45 to 57,        wherein introducing comprises transfecting.    -   Embodiment 59 is the method of any one of embodiments 45 to 58,        wherein introducing comprises electroporating, microinjecting,        cell squeezing, sonoporating, impalecting, or hydrodynamic        delivery.    -   Embodiment 60 is a method of making a cytoplast, the method        comprising:        -   transfecting a cell with a vector; and        -   enucleating the cell.    -   Embodiment 61 is the method of embodiment 60, wherein the        transfecting step precedes the enucleating step.    -   Embodiment 62 is the method of embodiment 61, wherein the        enucleating occurs after the vector integrates into the genome        of the cell.    -   Embodiment 63 is the method of embodiment 60, wherein the        enucleating step precedes the transfecting step.    -   Embodiment 64 is the method of any one of embodiments 60 to 63,        wherein the vector is a viral vector.    -   Embodiment 65 is the method of embodiment 64, wherein the viral        vector is a retrovirus vector, an adeno-associated virus (AAV)        vector, a vesicular virus vector, or a hybrid virus vector.    -   Embodiment 66 is the method of any one of embodiments 60 to 65,        wherein the vector comprises a coding sequence of a therapeutic        protein.    -   Embodiment 67 is the method of embodiment 66, wherein the        therapeutic protein is an enzyme, an antibody, an antigen, a        toxin, cytokine, a protein hormone, a growth factor, a cell        surface receptor, or a vaccine.    -   Embodiment 68 is a method of making a cytoplast comprising:        -   enucleating a cell.    -   Embodiment 69 is the method of embodiment 68, wherein the cell        is not an erythroblast.    -   Embodiment 70 is the method of embodiment 68 or embodiment 69,        wherein enucleating comprises centrifugation.    -   Embodiment 71 is a method of treating a subject comprising:        -   administering to the subject a therapeutically effective            amount of a cytoplast of any one of embodiments 33 to 44.    -   Embodiment 72 is a method of treating a subject comprising:        -   administering to the subject a therapeutically effective            amount of a cytoplast.    -   Embodiment 73 is the method of embodiment 72, wherein the        cytoplast is not obtained from an erythroblast.    -   Embodiment 74 is a method comprising:        -   making a cytoplast by the method of any one of embodiments            45 to 70; and        -   storing the cytoplast.    -   Embodiment 75 is the method of embodiment 74, wherein storing        comprises cryopreservation.    -   Embodiment 76 is the method of embodiment 74, wherein storing        comprises cryohibernation.    -   Embodiment 77 is a method comprising:        -   culturing cells in a media;        -   stimulating the cells; and        -   enucleating the cells to form cytoplasts.    -   Embodiment 78 is the method of embodiment 77, wherein culturing        comprises one or more of: 3D culturing, adherent culturing,        suspension culturing, and semi-suspension culturing.    -   Embodiment 79 is the method of any one of embodiments 77 to 78,        wherein stimulating the cells comprises one or more of: adding        one or more drugs to the media, adding one or more antibodies to        the media, adding one or more exosomes to the media, adding one        or more chemokines to the media, adding one or more cytoplasts        to the media, culturing under 2D or 3D conditions, or culturing        under hypoxic conditions.    -   Embodiment 80 is the method of any one of embodiments 77 to 80,        further comprising separating the cells or the cytoplasts from        the media.    -   Embodiment 81 is a method comprising:        -   culturing cells in a media; and        -   stimulating the cells, wherein stimulating the cells            comprises adding one or more cytoplasts to the media.    -   Embodiment 82 is the method of embodiment 81, wherein culturing        comprises one or more of: 3D culturing, adherent culturing,        suspension culturing, and semi-suspension culturing.    -   Embodiment 83 is the method of any one of embodiments 81 to 82,        wherein stimulating the cells further comprises one or more of:        adding one or more drugs to the media, adding one or more        antibodies to the media, adding one or more exosomes to the        media, adding one or more chemokines to the media, culturing        under 2D or 3D conditions, or culturing under hypoxic        conditions.    -   Embodiment 84 is the method of any one of embodiments 81 to 83,        further comprising separating the cells from the media.    -   Embodiment 85 is the method of any one of embodiments 81 to 84,        further comprising enucleating the cells to form cytoplasts.    -   Embodiment 86 is a method of treating a subject comprising:        -   administering a therapeutically effective amount of a media            prepared by the method of embodiment 80 or embodiment 84 to            the subject.    -   Embodiment 87 is a method of treating a subject comprising:        -   administering a therapeutically effective amount of the            cytoplasts prepared by the method of embodiment 80 or            embodiment 86 to the subject.    -   Embodiment 88 is use of the cytoplasts of any one of embodiments        33 to 44 in the manufacture of a medicament for the treatment of        a cancer, an infection, a neurological disease, a degenerative        disease, an autoimmune disease, a cardiovascular disease, an        ophthalmologic disease, a skeletal disease, a metabolic disease,        or two or more thereof    -   Embodiment 89 is use of a media prepared by the method of        embodiment 80 or embodiment 84 in the manufacture of a        medicament for the treatment of a cancer, an infection, a        neurological disease, a degenerative disease, an autoimmune        disease, a cardiovascular disease, an ophthalmologic disease, a        skeletal disease, a metabolic disease, or two or more thereof.    -   Embodiment 90 is a method of determining the presence or absence        of a disease or condition in a subject comprising: administering        cytoplasts to the subject.    -   Embodiment 91 is the method of embodiment 90, wherein the        disease or condition is a cancer, an infection, an inflammatory        condition, a neurological disease, a degenerative disease, an        autoimmune disease, a cardiovascular disease, an ischemic        disease, a genetic or inherited condition, a developmental        condition, an ophthalmologic disease, a skeletal disease, a        metabolic disease, a toxicosis, idiopathic disease, or two or        more thereof.    -   Embodiment 92 is the method of any one of embodiments 90 to 91,        wherein the cytoplasts express or contain a reporter molecule or        reagent.    -   Embodiment 93 is the method of embodiment 92, wherein the        reporter molecule or reagent is a bioreporter molecule or        reagent.    -   Embodiment 94 is a cell fusion product comprising:        -   a first cytoplast, wherein the first cytoplast is the            cytoplast of any one of embodiments 33-44; and        -   a cell or second cytoplast.    -   Embodiment 95 is a method of determining the presence or absence        of a disease or condition in a subject comprising:        -   obtaining a sample from a subject; and        -   adding cytoplasts to the sample.    -   Embodiment 96 is the method of embodiment 95, wherein the        disease or condition is a cancer, an infection, an inflammatory        condition, a neurological disease, a degenerative disease, an        autoimmune disease, a cardiovascular disease, an ischemic        disease, a genetic or inherited condition, a developmental        condition, an ophthalmologic disease, a skeletal disease, a        metabolic disease, a toxicosis, idiopathic disease, or two or        more thereof.    -   Embodiment 97 is the method of any one of embodiments 95 to 96,        wherein the cytoplasts express or contain a reporter molecule or        reagent.    -   Embodiment 98 is the method of embodiment 97, wherein the        reporter molecule or reagent is a bioreporter molecule or        reagent.

EXAMPLES

The disclosure is further described in the following examples, which donot limit the scope of the disclosure described in the claims.

Example 1—Successful Enucleation and Survival of Mammalian Cells

As shown in FIG. 1, therapeutic cytoplasts can be generated fromallogenic or autologous donor-derived cells, and can be used for diseasetreatment as well as for diagnostics. As a proof of concept, theenucleation efficiency and recovery rate of various types of mammaliancells (e.g., mesenchymal stem cells, neutrophils, fibroblast, andnatural killer cells) was determined. After removal of the mammaliancells from the cell culture plates, the mammalian cells were enucleatedby density gradient centrifugation using discontinuous Ficoll gradients,high-speed centrifugation (FIGS. 2A-D). Table 1 summarizes the resultsof enucleation using a suspension protocol. Enucleation efficiency andcell viability was the highest in both hTERT transformed and primarymesenchymal stem cells (MSCs), as well as in fibroblasts andneutrophils. Table 2 summarizes the results of enucleation using anadherent protocol. Enucleation efficiency was greater than 70% in bothmesenchymal stem cells and macrophages. This experiment showed thatvarious types of mammalian cells could undergo enucleation using any ofthe methods described herein.

TABLE 1 Enucleation efficiency and viability determinations of mammaliancells using the suspension protocol. Enu- Viability cleation Recoveryafter Yield Cell type Efficiency Rate 24 hours per run MSC cells AD-MSC90%-95% 60%-90% 80%-95% 12-15M (hTERT) UC-MSC 85%-90% 60%-80% 80%-95%10-15M (primary) BM-MSC 80%-90% 40%-50% 80%-90% ~8M (primary) NK cellsNKL 50%-85% 20%-50% 50%-75% ~8M NK-92 70%-90% 20%-40% 20%-40% ~5M Macro-RAW 85%-95% 40%-70% 20%-40% ~15M  phages 264.7 Neutrophils HL-60 60%-98%20%-40% 60%-80% ~15M  Fibroblasts L929 70%-90% 50%-70% 70%-90% ~15M NIH3T3 70%-80% 40%-50% 70%-80% ~9M Enucleation efficiency = enucleatedcells versus total recovered cells; Recovery rate = recovered cellsversus total input cells used for enucleation. Viability after 24 hours= live cells measured by Trypan blue staining versus total cells; Yieldper run = the number of cytoplasts harvested for each run; M = millioncells AD-MSC (hTERT) = human hTERT immortalized adipose-derivedmesenchymal stem cells; BM-MSC (primary) = human primary bonemarrow-derived mesenchymal stem cells; NK = natural killer cells.

TABLE 2 Enucleation efficiencies and viability determinations ofmammalian cells using the adherent protocol Viability EnucleationRecovery after Yield Cell type Efficiency Rate 24 hours per run MSCAD-MSC 70%-95% 40%-60% 80%-95% 1M cells (hTERT) Macro- RAW 264.7 85%-95%40%-70% 10%-30% ~1M  phages Enucleation efficiency = enucleated cellsversus total recovered cells; Recovery rate = recovered cells versustotal input cells used for enucleation. Viability after 24 hours = livecells measured by Trypan blue staining versus total cells; Yield per run= the number of cytoplasts harvested for each run; M = million cells

Next, the survival of cytoplasts was determined across 96 hours (FIG.3A). Whereas MSC proliferated over-time, cytoplasts did not. Instead,the relative fold change in viable cytoplasts remained fairly constantfor 72 hours before declining at 96 hours. Thus, cytoplast survivalspanned 3-4 days. As most cell-based therapies are not used immediately,the viability of cytoplasts after cryopreservation was determined.Surprisingly, the viability of cytoplast after cryopreservation wasgreater than the viability of MSC following cryopreservation (FIG. 3B).Cytoplasts plated immediately after enucleation and cytoplasts recoveredfrom cryopreservation displayed similar relative cell viability after 24hours (FIG. 3C). This experiment showed that cytoplasts survival was notaffected by cryopreservation.

Next, a large-scale production of cells was set up ex vivo, followed bylarge-capacity density gradient centrifugation and enucleation, whichlead to the generation of a therapeutic cytoplast. In one embodiment,the therapeutic cytoplast is loaded with therapeutic cargo (e.g., mRNA,drugs, peptides, etc. . . . ) for disease treatment. In anotherembodiment, the therapeutic cytoplast is prepared for immediate use(e.g., for intravenous injection (IV), intraperitoneal injection (IP),tissue, or in vitro applications) for diagnostic use.

Example 2—Cytoplasts Retain Intact Organelles, the Ability to Interactwith the Extracellular Matrix, Perform Cell-Biological Functions, andcan Serve as Delivery Vehicles with Therapeutic Value

After determining whether cytoplasts could retain viability aftercryopreservation, flow cytometry analysis were performed in order todetermine whether the cell surface marker profile of MSC-derivedcytoplasts differed from bone-marrow derived MSC (FIG. 4). As depictedin FIG. 4, both MSC-derived cytoplasts and bone-marrow derived MSCsmaintained cell surface expression of CD45, CD90, CD44, CD146, andCD166. FIGS. 5A-F′ and FIG. 6A-D′ showed that cytoplasts attached,reorganized the cytoskeleton, spread on matrix proteins in 2D and 3Dculture systems, and formed tunneling nanotubes, which can transferbioproducts between cells of the same or different origin.Organelle-staining indicated that Golgi, ER, F-actin cytoskeleton,lysosomes, endosomes, microtubules, and mitochondria remain intact incytoplasts (FIGS. 7A-E′). Furthermore, cytoplasts exhibited homingpotential in vitro. Cytoplasts readily migrated on extracellular matrixproteins and migrated directionally towards soluble chemokine gradients(i.e. via chemosensing) (FIGS. 8A and 8B). Notably, cytoplaststransfected exogenously with purified mRNAs produced functionalintracellular proteins, which could mimic therapeutic mRNA applicationsbeing developed for a variety of clinical uses and disease-states. Thisalso demonstrates that the machineries for mRNA translation and proteinsynthesis operate normally in cytoplasts in the absence of a nucleus,and thus can be used to produce bioactive molecules with therapeuticvalue.

Cytoplasts transfected exogenously with purified mRNA encoding knownsecreted proteins produce functional extracellular proteins inconditioned culture media, indicating that the ER/Golgi and secretorypathways operate normally in cytoplasts in the absence of a nucleus(FIG. 11). In addition, treatment of macrophages and endothelial cellswith cytoplast-conditioned media containing secreted proteins activatedkey signal transduction responses in these cells (FIG. 12). Thisprovided a proof of concept that cytoplasts could be used as novelvehicles to produce and deliver secreted proteins and biomolecules withtherapeutic value. Cytoplasts can be loaded with various cargoincluding, but not limited to, siRNA, shRNA, mRNA, DNA plasmids,peptides, and chemotherapeutic agents (see, e.g., FIGS. 9 and 10).

Example 3—Engineered Cytoplasts can Function Both In Vitro and In Vivo

Without wishing to be bound by theory, the examples show that cytoplaststhat have been engineered to express a “cargo”, e.g., an exogenous mRNAmolecule, can be produced. FIGS. 13B and 13C show that MSC-derivedcytoplasts can be engineered to produce and secrete therapeutic levelsof a functional anti-inflammatory cytokine interleukin 10 (IL-10) invitro and in a preclinical mouse model following intravenous injection.FIG. 13B shows that cytoplasts transfected with IL-10 mRNA can secretehigh levels of IL-10. To determine whether the secreted IL-10 is active,serum-starved macrophages were incubated with conditioned medium (CM)from untreated MSCs, MSCs expressing IL-10, untreated cytoplasts, andcytoplasts expressing IL-10. Phosphorylated STAT3 was detected inmacrophages following incubation with CM from MSCs expressing IL-10 andfollowing incubation with CM from cytoplasts expressing IL-10, whereasno STAT3 activity was detected in macrophages following incubation withCM from untreated MSCs and untreated cytoplasts (FIG. 13C). To determinewhether cytoplast-secreted IL-10 can be detected in vivo, C57Bl/6 micewere injected retro-orbitally with MSC or MSC-derived cytoplastsexpressing IL-10. Two hours post-injection, blood was collected and thelevels of IL-10 were determined. Little to no IL-10 was detected in theblood of mice that were injected with untreated MSC (FIG. 13D). As shownin FIG. 13D, higher levels of IL-10 were detected in mice injected withMSC-derived cytoplasts expressing IL-10 as compared to the level in miceinjected with untreated MSC.

These data illustrate the potential of genetically engineeredcytoplast-based cell therapies to produce and secreteclinically-relevant therapeutic cytokines to treat normal and diseasedtissues.

To determine whether MSC-derived cytoplasts can invade through thebasement membrane, MSC or MSC-derived cytoplasts were allowed to invadethrough the basement membrane towards 10% FBS for 24 hours. As shown inFIGS. 14A and 14B, MSC-derived cytoplasts were just was efficient atinvading the basement membrane as untreated MSCs in the presence of 10%FBS. Noteworthy, while untreated MSCs were able to invade the basementmembrane in the absence of a chemoattractant, MSC-treated cytoplastswere far less able to invade the basement membrane in the absence of achemoattractant. These data illustrate that MSC-derived cytoplasts candigest and invade through the basement membrane. These data illustratethe innate potential of cytoplast-based cell therapies to penetrate andmigrate through complex extracellular matrix barriers to deliver theircargo(s) within tissues.

As shown in FIGS. 15A and 15B, MSC-derived cytoplasts have an averagediameter of 12 μm, while MSC have an average diameter of 20 μm. Todetermine the biodistribution of MSC-derived cytoplasts, mice wereretro-orbitally injected with MSC or MSC-derived cytoplasts. As shown inFIGS. 15C and 15D, more MSC-derived cytoplasts were detected in theliver than the number of MSC detected in the liver. These dataillustrate the potential of cytoplast-based cell therapies to bedelivered directly to the circulation to treat a wide range of diseases.

Example 4—Engineered Cytoplasts can Express Functional Cell SurfaceProteins

As shown in FIG. 16B, engineered MSCs expressing CXCR4 and engineeredMSC-derived cytoplasts expressing CXCR4 express comparable levels ofCXCR4, as determined by flow cytometry. To determine whether engineeredcytoplasts can express functional cell surface proteins, MSCs andMSC-derived cytoplasts expressing CXCR4 receptors were allowed tomigrate towards various concentrations of SDF-1α. As shown in FIG. 16C,MSC-derived cytoplasts engineered to express functional CXCR4 canmigrate towards SDF-1α, and cell migration increases with increasingconcentrations of SDF-1α. Furthermore, the number of migratingMSC-derived cytoplasts was greater than the number of migrating MSCsexpressing CXCR4 (FIG. 16C).

FIGS. 17A-C show that MSC-derived cytoplasts can be engineered toexpress functional cell adhesion proteins known to mediate cell adhesionto the inflamed vasculature. FIGS. 18A-D show that MSC-derivedcytoplasts can be engineered to express cell proteins known to modulatemacrophage interactions and phagocytosis of therapeutic cells.

Example 5. Cytoplasts can be Engineered to Secrete Functional IL-12, andcan Induce the Expression of Inflammatory Genes and Suppress TumorGrowth in a Syngeneic Mouse Model of Breast Cancer

MSCs and MSC-derived cytoplasts were transfected with IL-12 mRNA.Conditioned medium (CM) was collected 24 hours, 48 hours and 72 hourspost-transfection. As shown in FIG. 19B, MSC-derived cytoplasts secreteIL-12. To determine whether MSC-derived cytoplasts can secretefunctional IL-12, mouse splenocytes were treated with full media, CMfrom MSC expressing IL-12, CM from MSC-derived cytoplasts expressingIL-12, and purified IL-12. MSC-derived cytoplasts expressing IL-12 andMSC expressing IL-12 secrete functional IL-12 that can causephosphorylation of STAT4 in mouse splenocytes (FIG. 19C).

As shown in Example 3, administration of cytoplasts retro-orbitally waswell tolerated in mice. To determine whether intratumoral administrationof cytoplasts was tolerated, mice were injected either by retro-orbitalor intratumoral administration. The number of deaths was recorded andclassified according to injection method and cause of death (Table 3).As shown in Table below, intratumoral administration of cytoplasts waswell-tolerated with an excellent safety profile.

TABLE 3 In vivo safety of cytoplast administration in mice InjectionNumber of Number of Method Animals Deaths Cause of Death CytoplastRetro-orbital 36 0 Intratumoral 113 1 Anesthesia-related

Next, MSC-derived cytoplasts expressing IL-12 and empty MSC-derivedcytoplasts were injected into established E0771 subcutaneous tumors.Forty-eight hours after injection, all mice were euthanized, and tumorsamples were collected. As shown in FIG. 19D, tumor IL-12 was detectedin tumors isolated from mice that were injected with MSC-derivedcytoplasts expressing IL-12, whereas little to no tumor IL-12 wasdetected in tumors isolated from mice that were injected with emptyMSC-derived cytoplasts. Taken together, these results indicate thatMSC-derived cytoplasts can produce, secrete and deliver clinicallyrelevant levels of therapeutic cytokines to a diseased tissue in apreclinical mouse model.

FIGS. 20A-C show that samples taken from mice injected with cytoplastsengineered to express IL-12 cytokine express interferon gamma (IFNγ),PD-L1 and CXCL9, whereas samples taken from mice that received only PBSor empty cytoplasts expressed low levels of IFNγ, PD-L1 and CXCL9. Thesedata indicate that MSC-derived cytoplasts engineered to express IL-12induced an inflammatory response within the injected tumor. FIG. 20Dshows a decrease in tumor size following injection of MSC-derivedcytoplasts engineered to express IL-12.

FIGS. 21A-C show that MSC-derived cytoplasts can be loaded withoncolytic viruses and can deliver such viruses to tumors growing inimmunocompromised and immunocompetent mice, which in combination withIL-12 secretion promotes infiltration of cytotoxic CD8+ T cells into thetumor. Regarding FIG. 21 A, it is notable that very few cytoplasts canbe detected in the tumor after 7 days, whereas a large number of MSCsare present in the center (injection site) and at the outer edge of thegrowing tumor.

FIGS. 22A-B show that genetically engineered MSC-derived cytoplasts candeliver gene editing proteins to regulate gene function in host cellsfollowing cytoplast-host cell fusion. These data illustrate thepotential of cytoplast-based cell therapies to deliver gene editingcomponents to modify normal or mutant genes in cells.

Enucleation of Mesenchymal Stem Cells (MSC)

This protocol was modified from Methods in Cell Biology Volume 14, 1976,Pages 87-93 Chapter 7 Enucleation of Mammalian Cells in Suspension(Michael H. Wigler, Alfred I. Neugut, I. Bernard Weinstein).

Preparation of 50% Ficoll solution: In a glass beaker shielded fromlight, grams of Ficoll (PM400, GE Healthcare 17-0300-500) were dissolvedin an equivalent number of milliliters ultrapure water (Invitrogen10977-015) by continual magnetic stirring for 24 hours at roomtemperature. The mixture was then autoclaved for 30 minutes. Once themixture was cooled, it was stirred again to ensure uniform consistency.The refractive index was measured on a refractometer (Reichert13940000), and was in the range of 1.4230-1.4290. Aliquots were storedat −20 degrees Celsius.

Preparation of 2×MEM: For each 50 ml quantity, 10 mL 10×MEM (Gibco,11430-030), 2.94 mL exactly Sodium Bicarbonate (7.5%, Gibco, 25080-094),1 mL 100× Pen-Strep (Gibco 15140-122) and 36 mL ultrapure water(Invitrogen 10977-015) was used. The solution was then filtered through0.22 um membrane flask (Olympus 25-227) and stored at 4 degrees Celsius.

On the day before enucleation, MSCs were seeded at 2.5 M per 15 cm plate(Olympus 25-203) in 20 mL MSC medium [MEM 1× (Gibco 12561-056); 16.5%premium FBS (Atlanta Biologics S1150); 1% HEPES 1M (Gibco 15630-80); 1%Anti-Anti 100× (Gibco 15240-062); 1% Glutamax 100× (Gibco 35050-061)].Next, Cytochalasin B (Sigma Aldrich C6762) was added to the 2×MEM (2μM/mL final concentration).

Preparation of Ficoll gradients: 2×CytoB was added to 50% Ficollaliquots at 1:1 dilution to make 25% Ficoll stock concentration. Next,17%, 16%, 15% and 12.5% Ficoll were made by diluting 25% Ficoll with theappropriate volume of 1×MEM buffer (2×MEM containing Cytochalasin Badded to ultrapure water at 1:1 dilution). The dilutions wereequilibrated in a CO2 incubator for at least 1 hour covered with loosecap. The Ficoll gradients were then poured into 13.2 mL ultra-cleartubes (Beckman, 344059), and incubated overnight (6-18 hours) in the CO2incubator.

On the day of enucleation, 12-25M MSC (ideally 20M) were collected intoeach tube for enucleation. Media was aspirated, and the cells washedonce with phosphate buffered saline (PBS) (GIBCO 14190-144). Five mL ofTrypLE-Select (Gibco, 12563011) was added to each plate, and incubatedup to 5 minutes. When 90% of the cells were detached, 5 mL full MSCmedia was added, and the cells were collected into 50 ml tubes (3-4plates/tube). The tubes were then centrifuged at 1, 200 rpm for 5minutes. The pellet was resuspended in 10 mL PBS. Cells were counted,pelleted, and re-suspended with 12.5% Ficoll. Next, the cell-Ficollmixture was dropwise passed through a 40 um cell strainer (Falcon352340) into a new 50 mL tube. Using a syringe, 3.2 mL of cellsuspension was slowly loaded onto the pre-made gradients. One mL of1×MEM buffer was added at the final (top) layer with syringe. The tubeswere then loaded into rotor buckets, balanced, and run in theultracentrifuge (Beckman, L8M) for 60 minutes, 26,000 rpm, 31° C., Accel7, Deccel 7. At the end of the centrifugation, there were three layers:one near the top of the 12.5% (cytoplasts and debris), one near the12.5/15% interface (cytoplasts), and a pellet at the bottom of the 25%(karyoplasts). The layers above 15% Ficoll solution were collected into15 ml conical tubes. The collected layers are then diluted with morethan 4 volumes warm serum-free MSC medium (i.e. 3 mL of Ficoll andfilled with up to 15 mL media). After gently mixing, the mixture waspelleted for 10 minutes at 1,200 rpm. Following three washes with warmserum-free MSC medium, the cells were resuspended in media according tothe experimental protocol, e.g., transfection media vs. migration mediavs. serum free media vs. full media. Efficiency of enucleation wasdetermined in a 12-well plate by adding full MSC media with 1:2000dilution Vybrant® Dyecycle™ Green (Molecular Probes V35004) or 1:5000dilution Hoechst 33342. A small volume of each layer was added to eachwell and allowed to attach/stain for 10 minutes in the incubator. Thepercentage of negative cytoplasts per population was determined byepifluorescent microscopy.

Cytoplast mRNA Transfection

1 M cytoplasts were suspended with warm 1 ml amino acid-free α-MEM fullmedium (ThermoFisher 12561056; 16.5% Premium fetal bovine serum (FBS),1% Glutamax (Gibco 35050061), 1% HEPES (Gibco 15630080)). 1 μg mRNA wasdiluted with warm opti-MEM and mixed with pipet at least 20 times. 4 μllipofectamine-3000 (ThermoFisher L300015) was added to 46 μl warmopti-MEM (ThermoFisher 31985062) and mixed with pipet for at least 20times. The ratio of mRNA and lipofectamine-3000 was 1:4 (w/v). The mRNAand lipofectamine-3000 dilutions were mixed with pipet for at least 20times and incubated at room temperature for 15 minutes. The mRNA andlipofectamine-3000 mixture was added to the cytoplast suspension, mixedwell and incubated at 37° C. for 30 minutes. The suspension was shakenevery 5 minutes to prevent cell clumping. After incubation, the cellswere centrifuged, and re-suspended in normal α-MEM full medium (16.5%Premium FBS, 1% Antibiotic-Antimycotic, 1% Glutamax, 1% HEPES) or PBS.

Cytoplast siRNA Transfection

1 M cytoplasts were suspended with warm 1 ml A/A free α-MEM full medium(16.5% Premium FBS, 1% Glutamax, 1% HEPES). Two μl siRNA was dilutedwith warm opti-MEM and mixed with pipet at least 20 times. Eight μllipofectamine-3000 was diluted with 92 μl warm opti-MEM and mixed withpipet at least 20 times. The ratio of siRNA and lipofectamine-3000 was1:4 (v/v). The siRNA and lipofectamine-3000 dilutions were mixed withpipet at least 20 times and incubated at room temperature for 15minutes. The siRNA and lipofectamine-3000 mixture was added to thecytoplast suspension, mixed well and incubated at 37° C. for 20 minutes.The suspension was shaken every 5 minutes to prevent cell clumping.After a 20 minute incubation, the cells were centrifuged, andre-suspended with normal α-MEM full medium (16.5% Premium FBS, 1%Antibiotic-Antimycotic, 1% Glutamax, 1% HEPES).

Generation of Oncolytic Virus Infected Cytoplasts

One day before enucleation (usually 18 hrs before enucleation),2.5*10{circumflex over ( )}6 hTERT-MSCs were seeded on a 15-cm dish.Roughly two hours after seeding, the cells were washed once with PBS.Cells were then infected with oHSV-GFP (Imams OV3001) at different MOIs(0.05 or 0.5 for example) with 8 mL serum free opti-MEM. Next, cellswere incubated at 37° C. for 2 hours with occasionally shaking. Thevirus inoculum was then discarded. 20 mL pre-warmed full culture medium(α-MEM, 16.5% Premium FBS, 1% Antibiotic-Antimycotic, 1% Glutamax, 1%HEPES) was added to each well. The cells were incubated at 37° C. untilenucleation.

Lentivirus Overexpressing Functional Proteins in Cytoplasts

Target cells were plated in one well of 6-well plate at density of1-2×10⁵ cells/well, or 10 cm plate with 0.5-1 M MSCs. The next day, theconcentrated recombinant lentivirus was thawed in a 37° C. water bathand removed from the bath immediately once thawed. The cells were thenwashed with PBS 3 times. 200 μL serum free medium or 2 mL serum freemedium (1:1250 SureENTRY) was added. The target cells were infected in a6-well plate with MOI 10:1. The next day, the viral supernatant wasremoved and the appropriate complete growth medium was added to thecells. After 72 hours incubation, the cells were subcultured into 2×100mm dishes. The appropriate amount of selection drug (i.e. puromycin) wasadded for stable cell-line generation. 10-15 days after selection,clones were picked for expansion and were screened for positive ones.The selected positive clones were expanded for enucleation. Engineeredcytoplasts were prepared as outlined above. The target proteinexpression on cytoplasts was determined by ordinary biochemical methodsor functional assays, e.g., fluorescent activated cell sorting (FACS),western blot, or Boyden chamber assay.

Peptide Loading into Cytoplasts

1×10⁵/ml per well were plated onto a 4-chamber glass slide (LabTek II4-chamber glass slide, 155383) in full MSC media [MEM 1× (Gibco12561-056); 16.5% premium FBS (Atlanta Biologics S1150); 1% HEPES 1M(Gibco 15630-80); 1% Anti-Anti 100× (Gibco 15240-062); 1% Glutamax 100×(Gibco 35050-061)]. Cells were allowed to attach for at least 1 hour orovernight. Cells were then rinsed with PBS (Gibco 14190-144). Arg9(FAM)(10 mM, Anaspec, AS-61207) was diluted in full media to a totalconcentration of 1:100 (100 uM). Cytoplasts were then incubated for 1 to2 hours, and rinsed 3 times with PBS. Hoechst 33342 (Invitrogen) wasadded at a 1:5000 dilution in full media for at least 10 minutes. Cellswere then washed with PBS and imaged by epifluorescent microscopy.

Generation of Pre-Clinical Syngeneic Tumor Model in Immune CompetentMice

A small patch of fur on each side of the mouse's flank (from the levelof the elbow to above the thigh and just onto the abdomen to halfwayacross the back) was shaved. Excess fur was wiped away with an alcoholwipe. Using 1 mL tuberculin syringe with 27G ½″ needle, 1M/100 μL E0771cells were injected into each side of the mouse. Mice were monitoreduntil tumors reached 0.7-1.0 cm diameter, roughly 10-20 days later.

Intratumoral Delivery of Therapeutic Cytoplasts

Engineered cytoplasts (i.e. loaded with IL-12 mRNA) were resuspended inPBS at desired concentration, e.g., 3M/50 uL. On the day of injection,animal weight and tumor dimensions were measured. Engineered cytoplastswere injected into the center of the tumor. Mice were monitored; tumorsand body weight were measured every 2-3 days.

Intravenous Delivery of Therapeutic Cytoplasts

Engineered cytoplasts (i.e. loaded with IL-12 mRNA) were resuspended inPBS at desired concentration, e.g., 3M/50 μL. The maximum recommendedinjection volume was 100 μL. Injections were performed with a 1 mLtuberculin syringe and 27G ½″ or 28G ½″ needle. Institutional AnimalCare and Use Committee (IACUC) protocols were followed for intravenous(IV) injection. Retro-orbital injections were performed under anesthesiawith ketamine/xylazine intraperitoneal (IP) injection or isofluoraneinhalation. Tail vein injections were performed using a restraintdevice.

Example 6. 3D-Cultured MSC can be Enucleated and 3D-Derived CytoplastsShow Better Biodistribution In Vivo

MSCs were cultured in 3D-hanging drops (3D MSCs) then enucleated togenerate 3D cytoplasts. The 3D culture protocol of MSC by hanging dropsis modified from Curr Protoc Stem Cell Biol. 2014 Feb. 6; 28: Unit-2B.6.(Thomas J. Bartoshl and Joni H. Ylostalo).

Healthy MSCs were harvested from 2D-cultured plates by Trypsin andresuspended in fresh α-MEM (ThermoFisher 12561056) full medium (16.5%Premium FBS, 1% Antibiotic-Antimycotic, 1% Glutamax, 1% HEPES) at 1.43million cells/ml. The lid of a 15 cm plate was opened completely and 20ml PBS was added to the plate. A multichannel pipette was used to makedroplets on the lid of the plate at 35 μl per droplet (approx. 50,000cells/droplet). About 100-120 droplets were placed on each lid. The lidwas closed and the plate was placed back into the incubator. Dropletswere cultured for 2 days, then harvested by cell lifter and collectedinto 15 ml tubes (approx. 300 droplets per tube). The tubes werecentrifuged for 5 minutes at 1,200 rpm. The supernatant was removed andthe tubes were washed twice with PBS. All P BS was then removed and 7.5ml of freshly thawed 0.25% Trypsin-EDTA (ThermoFisher 25200114) wasadded to each tube. The tubes were incubated in a water bath for 4minutes. The droplets were gently pipetted with 1 ml pipettes withlow-retention tips about 10-20 times and incubated in the water bath foranother 4 minutes. The droplets were again gently pipetted with 1 mlpipettes with low-retention tips about 10-20 times until most of thedroplets were dissociated. 7.5 ml of full serum medium (GlutaMAXSupplement (Gibco 35050061); Fetal Bovine Serum—Premium Select (AtlantaBiologicals S11550); HEPES (1 M) (Gibco 15630080);antibiotic-Antimycotic (100×) (Gibco 15240062)) was added to each tubeand the tubes were centrifuged for 10 minutes at 1,200 rpm. Thedissociated cells were washed with 10 ml of full serum medium and thecells were resuspended with 5 ml full serum medium. The cells werepassed through a 70 μm cell filter and then the filter was washed with 5ml full serum medium. The cells were counted and resuspended withpre-treated 12.5% Ficoll at more than 10M/ml. 30-40M cells were used foreach enucleation tube. Subsequently, the protocol for enucleationdescribed above was followed.

DiD labeled normal 2D-cultured MSCs (2D MSC), 3D MSCs or 3D cytoplastswere retro-orbitally injected into BalB/C mice respectively. Indicatedtissues were harvested 24 hours after injection and DiD labeled cellsanalyzed by FACS. FIG. 24 shows the successful generation of 3D-derivedcytoplasts from 3D-cultured MSCs and also shows the 3D-derivedcytoplasts have less lung trapping and better biodistribution toperipheral organs than 2D-cultured cells after injection into thecirculation. This is expected to greatly improve their therapeuticability to locate and deliver cargo to tissues.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1.-20. (canceled)
 21. A cell fusion partner comprising: a cell without anucleus comprising: (a) an exogeneous nucleic acid molecule encoding afusion protein specific to cluster of differentiation 8 (CD8); and (b)an intracellular organelle sufficient for in vivo expression of thefusion protein in absence of the nucleus.
 22. The cell fusion partner ofclaim 21, wherein the cell without the nucleus further comprises asecond exogenous nucleic acid molecule encoding a therapeutic agentexpressed by the intracellular organelle in the absence of the nucleus.23. The cell fusion partner of claim 22, wherein the therapeutic agentcomprises a chimeric antigen receptor (CAR).
 24. The cell fusion partnerof claim 21, wherein the exogenous nucleic acid molecule comprises aviral nucleic acid molecule.
 25. The cell fusion partner of claim 22,wherein the second exogenous nucleic acid molecule comprises messengerRNA (mRNA)
 26. The cell fusion partner of claim 21, wherein the fusionprotein is a paramyxovirus virus protein or fragment thereof.
 27. Thecell fusion partner of claim 21, wherein the cell without the nucleushas a diameter comprising less than or equal to about 100 micrometers.28. A pharmaceutical formulation comprising: (a) the cell fusion partnerof claim 21; and (b) a pharmaceutically acceptable: excipient, diluent,or carrier.
 29. The pharmaceutical formulation of claim 28, formulatedfor human administration for the treatment of non-Hodgkin lymphoma(NHL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia(ALL), multiple myeloma (MM), or a combination thereof.
 30. The cellfusion partner of claim 21, that is isolated.
 31. A cell fusion productcomprising: (a) a cell without a nucleus comprising: (i) an exogeneousnucleic acid molecule encoding a fusion protein specific to CD8; and(ii) an intracellular organelle sufficient for in vivo expression of thefusion protein in absence of the nucleus; and (b) a target cellexpressing CD8 that is bound to the fusion protein expressed by the cellwithout the nucleus.
 32. The cell fusion product of claim 31, whereinthe target cell is a cancer cell.
 33. The cell fusion product of claim33, wherein the cancer cell is a cell from a non-Hodgkin lymphoma (NHL),a chronic lymphocytic leukemia (CLL), a acute lymphoblastic leukemia(ALL), a multiple myeloma (MM), or a combination thereof.
 34. The cellfusion product of claim 31, wherein the cell without the nucleus furthercomprises a second exogenous nucleic acid molecule encoding atherapeutic agent expressed by the intracellular organelle in theabsence of the nucleus.
 35. The cell fusion product of claim 34, whereinthe therapeutic agent comprises a chimeric antigen receptor (CAR). 36.The cell fusion product of claim 31, wherein the exogenous nucleic acidmolecule comprises a viral nucleic acid molecule.
 37. The cell fusionproduct of claim 32, wherein the second exogenous nucleic acid moleculecomprises messenger RNA (mRNA).
 38. The cell fusion product of claim 31,wherein the fusion protein is a paramyxovirus virus protein or fragmentthereof.
 39. The cell fusion product of claim 31, wherein the cellfusion product has a lifespan in a subject comprising fewer than orequal to about five days.
 40. The cell fusion product of claim 31,wherein the cell without the nucleus has a diameter comprising less thanor equal to about 100 micrometers.